Cancer cell-specific apoptosis-inducing agents that target chromosome stabilization-associated genes

ABSTRACT

The present inventors discovered that inhibition of the expression of various genes associated with chromosome stabilization induces cancer cell-specific apoptosis and inhibits cell proliferation. Compounds that inhibit expression of a gene associated with chromosome stabilization or inhibit the function of a protein encoded by such a gene are thought to have cancer cell-specific apoptosis-inducing effects.

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TECHNICAL FIELD

The present invention relates to cancer cell-specific apoptosis-inducingagents that target chromosome stabilization-associated genes and methodsof screening for the apoptosis-inducing agents.

BACKGROUND ART

Chromosomes are maintained in a stable state within cells by the actionof various cellular functions (genes). Examples of typical cellularfunctions (genes) that contribute to this chromosome stabilization areas follows:

(a) Genes Associated with Human Chromosomal Instability Disorders

Chromosome breakage, deletion, translocation, and aneuploidy areobserved in cells from patients with human chromosomal instabilitydisorders, and these cells are also sensitive to DNA damage-inducingdrugs. The occurrence of such instabilities indicates that humanchromosomal instability disorder-associated genes are involved inchromosome stabilization.

(b) Chromosomal DNA Replication Reaction Including Initiation ofChromosomal DNA Replication and Progression of Replication Fork

The chromosomal DNA replication reaction plays the role of replicatingchromosomal DNA during cell proliferation. It has the function ofmaintaining the number of chromosomes by accurately doubling thechromosomes when a cell divides into two cells.

(c) DNA Damage Checkpoints

DNA damage checkpoints play the role of checking for DNA damage,including breakage, chemical modification, and crosslinking, inchromosomes when the cell cycle advances from each of G1, S, G2, and Mphases to the next phase. These checkpoints have the function ofremoving chromosomal DNA damage before proceeding to the next stage ofthe cell cycle.

(d) Sister Chromatid Agglutination and Separation

Sister chromatid agglutination and separation play the role ofaccurately separating, into daughter cells, sister chromatids in somaticcells in which replication has been completed.

(e) Base Excision Repair

Base excision repair plays the role of removing modified bases when achemical modification damage, including oxidation and methylation, hasoccurred in bases in chromosomal DNA.

(f) Mismatch Excision Repair

Mismatch excision repair plays the role of recognizing mismatched basepairs other than the correct G-C and A-T base pairs present inchromosomal DNA, and repairing them to the correct base pairs.

(g) Nucleotide Excision Repair

Nucleotide excision repair plays the role of repairing DNA byrecognizing and removing DNA damage such as cyclobutane pyrimidinedimers and 6-4 photoproducts, which occur in chromosomal DNA due toultraviolet irradiation, and DNA internal crosslinking, which occursbetween adjacent bases in chromosomal DNA due to cisplatin.

(h) Homologous Recombination Repair

Using an undamaged homologous chromosome as a template, homologousrecombination repair plays the role of repairing various DNA damage,including breaks and gaps occurring in chromosomal DNA, and DNA damageresulting from incomplete repair by mechanisms such as base excisionrepair, mismatch excision repair, and nucleotide excision repair.

(i) Non-Homologous End-Joining Repair (Non-Homologous RecombinationRepair)

Non-homologous end-joining repair (non-homologous recombination repair)plays the role of repairing double-strand breaks in chromosomal DNA byjoining the ends.

(j) Double-Strand DNA Break Repair

Double-strand DNA break repair plays the role of repairing double-strandbreaks occurring in chromosomal DNA. This repair mechanism includeshomologous recombination repair and non-homologous end-joining repair(non-homologous recombination repair).

(k) DNA Post-Replication Repair (DNA Damage Tolerance)

DNA post-replication repair (DNA damage tolerance) is a mechanism thatenables repair of a damaged DNA strand when damaged chromosomal DNA isreplicated. Residual DNA damage is repaired following replication bythis mechanism.

(l) DNA Crosslink Damage Repair

DNA crosslink damage repair plays the role of repairing DNA crosslinkdamage within and between chromosomes caused by crosslinking agents suchas cisplatin.

(m) DNA-Protein Crosslink Damage Repair

DNA-protein crosslink damage repair plays the role of removingcovalently bonded complexes and crosslinked complexes when a covalentlybonded enzyme protein-DNA complex, which is a reaction intermediate ofDNA repair, has been formed, or a crosslinked complex between a base inchromosomal DNA and a protein has formed.

(n) DNA Polymerase

DNA polymerases play the role of carrying out DNA synthesis reactions inchromosome stabilization mechanisms such as replication, recombination,and repair.

(o) Nuclease

Nucleases play the role of decomposing DNA in chromosome stabilizationmechanisms such as replication, recombination, and repair.

(p) Nucleotide Cleansing

Nucleotide cleansing plays the role of removing modified bases whenchemical modification damage, including oxidation and methylation, hasoccurred in a base of a nucleotide serving as the substrate of a DNAsynthesis reaction.

(q) Chromatin Structure Maintenance

Chromatin structure maintenance plays a role in chromosome stabilizationmechanisms such as replication, recombination, and repair, throughmaintaining the higher order chromosomal structure.

(r) Telomere Structure Maintenance

Telomere structure maintenance plays an important role in chromosomestabilization via the control of chromosome end telomere length and theformation and maintenance of special higher order structures in telomereregions.

In addition, various genes related to the aforementioned functions havebeen reported to be involved in chromosome stabilization. For example,various findings have been reported regarding various genes involved inchromosome stabilization (see Non-Patent Documents 1 to 83).

However, the correlation between the aforementioned functions (genes)involved in chromosome stabilization and the induction of cancer-cellspecific apoptosis was so far unknown.

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[Non-patent Document 80] Budd, M. E., Choe, W. C. and Campbell, J. L.(1995). DNA2 encodes a DNA helicase essential for replication ofeukaryotic chromosomes. J Biol Chem., 270, 26766-26769.

[Non-patent Document 81] Budd, M. E. and Campbell, J. L. (1995). A yeastgene required for DNA replication encodes a protein with homology to DNAhelicases. Proc Natl Acad Sci USA., 92, 7642-7646.

[Non-patent Document 82] Tang, J. and Chu, G (2002). Xerodermapigmentosum complementation group E and UV-damaged DNA-binding protein.DNA Repair (Amst)., 1, 601-616.

[Non-patent Document 83] Martin-Lluesma, S., Stucke, V. M. and Nigg, E.A. (2002). Role of Hec1 in spindle checkpoint signaling and kinetochorerecruitment of Mad1/Mad2. Science., 297, 2267-2270.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to provide cancer cell-specificapoptosis-inducing agents. More specifically, an objective of thepresent invention is to provide cancer cell-specific apoptosis-inducingagents having as an active ingredient a compound which inhibitschromosome stabilization, a compound which inhibits expression of a geneinvolved in chromosome stabilization, or a compound which inhibits afunction of a protein encoded by the gene, and methods of screening forthe apoptosis-inducing agents. Another objective of the presentinvention is to provide methods for producing an apoptosis-inducingagent as a pharmaceutical composition.

To achieve the above objectives, the present inventors examined whetherapoptosis is induced cancer cell-specifically by abnormalities invarious functions relating to chromosome stabilization in cells. Thefollowing functions that are deeply involved in chromosome stabilizationwere selected as cellular chromosome stabilization-associated functions:(a) genes associated with human chromosomal instability disorders, (b)chromosomal DNA replication reaction including initiation of chromosomalDNA replication and progression of replication fork, (c) DNA damagecheckpoints, (d) sister chromatid agglutination and separation, (e) baseexcision repair, (f) mismatch excision repair, (g) nucleotide excisionrepair, (h) homologous recombination repair, (i) non-homologousend-joining repair (non-homologous recombination repair), (j)double-strand DNA break repair, (k) DNA post-replication repair (DNAdamage tolerance), (l) DNA crosslink damage repair, (m) DNA-proteincrosslink damage repair, (n) DNA polymerase, (o) nuclease, (p)nucleotide cleansing, (q) chromatin structure maintenance, and (r)telomere structure maintenance.

The present inventors examined the cancer cell apoptosis-inducingeffects of various genes involved in each of the aforementionedfunctions using siRNA having expression inhibitory effects on the genes.As a result, it was found that apoptosis was induced in cancer cellswhen the expression of a plurality of genes involved in each of theaforementioned functions were inhibited, and that this brought about aninhibition of cancer cell proliferation. The present inventors alsodiscovered that induction of apoptosis does not occur with respect tonormal cells (wild-type cells) even if the expression of these geneswere inhibited. These genes are considered to be target molecules forpreparing highly superior anticancer agents (carcinostatics) having fewadverse side effects.

The above results suggested that inhibition of the expression of genesinvolved in each of the aforementioned functions would be able to induceapoptosis. In addition, these genes are deeply involved in each of theaforementioned functions, and inhibition of the expression of the genesgenerally prevents the functions from working normally in cells. Thus,the aforementioned findings made by the present inventors indicates noneother than the fact that cancer cell-specific apoptosis is induced todue to abnormalities in each of the aforementioned functions.Accordingly, compounds that inhibit the aforementioned functions areconsidered to have the action of inducing cancer cell-specificapoptosis.

In addition, abnormalities in the aforementioned functions are known todestabilize chromosomes. Thus, chromosome destabilization in cells isconsidered to trigger induction of cancer cell-specific apoptosis.Namely, compounds that inhibit chromosome stabilization in cells, orcompounds that inhibit the function of genes involved in chromosomestabilization, are expected to serve as cancer cell-specificapoptosis-inducing agents.

The present invention provides cancer cell-specific apoptosis-inducingagents having as an active ingredient a compound which inhibitschromosome stabilization, a compound which inhibits expression of a geneinvolved in chromosome stabilization, or a compound which inhibits thefunction of a protein encoded by said gene, and methods of screening forsaid apoptosis-inducing agents. More specifically, the present inventionprovides the following:

[1] a cancer cell-specific apoptosis-inducing agent, comprising acompound that inhibits chromosome stabilization;

[2] the apoptosis-inducing agent of [1], wherein inhibition ofchromosome stabilization is due to the inhibition of any one of thefollowing functions (a) to (r):

(a) genes associated with human chromosomal instability disorders,

(b) chromosomal DNA replication reaction including initiation ofchromosomal DNA replication and progression of replication fork,

(c) DNA damage checkpoints,

(d) sister chromatid agglutination and separation,

(e) base excision repair,

(f) mismatch excision repair,

(g) nucleotide excision repair,

(h) homologous recombination repair,

(i) non-homologous end-joining repair (non-homologous recombinationrepair),

(j) double-strand DNA break repair,

(k) DNA post-replication repair (DNA damage tolerance),

(l) DNA crosslink damage repair,

(m) DNA-protein crosslink damage repair,

(n) DNA polymerases,

(O) nucleases,

(p) nucleotide cleansing,

(q) chromatin structure maintenance, and

(r) telomere structure maintenance;

[3] a cancer cell-specific apoptosis-inducing agent, comprising acompound that inhibits expression of a gene involved in any one of thefunctions of (a) to (r) described in [2];

[4] a cancer cell-specific apoptosis-inducing agent, comprising as anactive ingredient a compound that inhibits expression of any one of thefollowing genes:

APE2, ATR, BRCA1, Chk1, Cdc5, Cdc6, Cdc7, Cdc45, Cdt1, CSA, CSB, Ctf18,DDB1, DDB2, DNA2, DUT, Elg1, EndoV, Esp1, Exonuclease1, FBH1, FEN1,Geminin, Hus1, KNTC2 (NDC80), Ku80, Ligase1, Mad2, MBD4, Mcm3, Mcm4,Mcm5, Mcm6, Mcm7, Mcm8, Mcm10, MGMT, MLH3, Mms4, MPG, MSH2, Mus81, NBS1,NEIL2, NEIL3, NTH1, Orc1, Orc3, PARP1, PCNA, Pif1, PMS1, PMS2, PNK, Polap180, Pola p70, Pola Spp1 (Prim2a), Polb, Pold p125, Pole Dpb3, PoleDpb4, Pole Pol2, Poli, Poll, Polm, Psf1, Psf2, Psf3, Rad1, Rad18,Rad23A, Rad23B, Rad51, Rad51D, Rad54, Rad6A, RPA34, RPA70, Scc1, Scc3,Sir2, SIRT1 (Sirtuin), TDG, TDP1, TIMELESS, Tin2, Topoisomerase I,Topoisomerase IIIa, Topoisomerase IIIb, Ubc13, UNG, XAB2, XPC, XPF, XPG,Xrcc2, and XRCC4;[5] the apoptosis-inducing agent of [4], wherein nucleotide sequence ofeach gene described in [4] is selected from the group consisting of thenucleotide sequences described in SEQ ID NOs: 1 to 637 and 810 to 908;[6] the apoptosis-inducing agent of [4], wherein the compound thatinhibits expression of any one of the genes described in [4] is adouble-strand RNA having an RNAi effect (siRNA) on said gene;[7] the apoptosis-inducing agent of [6], wherein the double-strand RNAis a double-strand RNA comprising a sense RNA consisting of a sequencehomologous with arbitrary 20 to 30 contiguous bases in an mRNA of anyone of the genes described in [4], and an antisense RNA consisting of asequence complementary to said sense RNA,[7b] the apoptosis-inducing agent of [6], wherein the double-strand RNAhaving an RNAi effect is a double-strand RNA in which one strand of thedouble strand is a nucleotide sequence described in SEQ ID NOs: 724 to809 (this strand of the double strand is composed of a region excludingthe terminal TT from the sequence) or a nucleotide sequence described inSEQ ID NOs: 974 to 1063, and the other strand is a nucleotide sequencecomplementary to said nucleotide sequence,[7c] the apoptosis-inducing agent of [6], wherein the double-strand RNAhaving an RNAi effect is a double-strand RNA in which one strand of thedouble strand is a nucleotide sequence with one or a small number ofnucleotide additions, deletions, or substitutions to a nucleotidesequence described in SEQ ID NOs: 724 to 809 (this strand of the doublestrand is composed of a region excluding the terminal TT from thesequence) or SEQ ID NOs: 974 to 1063, and the other strand is anucleotide sequence complementary to said nucleotide sequence, whereinthe double-strand RNA has a function to inhibit expression of any of thegenes described in [4] above,[7d] the cancer cell-specific apoptosis-inducing agent comprising as anactive ingredient a molecule having a structure in which one end of thedouble-strand RNA is closed (forming a hairpin),[8] a cancer cell-specific apoptosis-inducing agent, comprising as anactive ingredient a DNA able to express a double-strand RNA having anRNAi effect on any one of the genes described in [4];[9] the apoptosis-inducing agent of [4], wherein the compound thatinhibits expression of any one of the genes described in [4] is thefollowing (a) or (b):(a) an antisense nucleic acid against a transcription product of saidgene or a portion thereof, or(b) a nucleic acid having ribozyme activity which specifically cleaves atranscription product of said gene;[10] a cancer cell-specific apoptosis-inducing agent comprising as anactive ingredient a compound that inhibits the function of a proteinencoded by any one of the genes described in [4];[11] the apoptosis-inducting agent of [10], wherein the compound thatinhibits the function of a protein encoded by any one of the genesdescribed in [4] is a compound of any one of the following (a) to (c):(a) a mutant protein having a dominant negative trait with respect to aprotein encoded by said gene;(b) an antibody which binds to a protein encoded by said gene; and,(c) a low molecular weight compound that binds to a protein encoded bysaid gene;[12] an anticancer agent, comprising as an active ingredient anapoptosis-inducing agent of any one of [1] to [11];[13] a method of screening for a cancer cell-specific apoptosis-inducingagent, comprising the following steps (a) to (c):(a) contacting a test compound with a protein encoded by any one of thegenes described in [4], or a partial peptide of the protein;(b) measuring the binding activity between the protein, or partialpeptide thereof, and the test compound; and(c) selecting a compound which binds to the protein encoded by saidgene, or the partial peptide of the protein;[14] a method of screening for a cancer cell-specific apoptosis-inducingagent, comprising the following steps (a) to (c):(a) contacting a test compound with a cell that expresses any one of thegenes described in [4], or a cell extract thereof;(b) measuring the expression level of said gene; and(c) selecting a compound which lowers said expression level as comparedto a level measured in the absence of the test compound;[15] a method of screening for a cancer cell-specific apoptosis-inducingagent, comprising the following steps (a) to (c):(a) contacting a test compound with a cell comprising a DNA having astructure in which the transcriptional regulatory region of any one ofthe genes described in [4] is operably linked to a reporter gene, orwith a cell extract thereof;(b) measuring the expression level of the reporter gene; and(c) selecting a compound which lowers the expression level as comparedto a level measured in the absence of the test compound;[16] a method of screening for a cancer cell-specific apoptosis-inducingagent, comprising the following steps (a) to (c):(a) contacting a test compound with a protein encoded by any one of thegenes described in [4], or a cell that expresses said protein, or a cellextract thereof;(b) measuring the activity of the protein; and(c) selecting a compound which lowers the activity of the protein ascompared to an activity measured in the absence of the test compound;and[17] a method for producing the apoptosis-inducing agent of [4] or [10]as a pharmaceutical composition, comprising the following steps (a) and(b):(a) screening for a compound by a method of any one of [13] to [16]; and(b) mixing said compound with a pharmaceutically acceptable carrier.

In addition, a specific embodiment of the present invention provides acancer cell-specific apoptosis-inducing agent containing as its activeingredient an siRNA molecule having as one of the strands of thedouble-strand RNA a nucleotide sequence described in any of SEQ ID NOs:724 to 809 and 974 to 1063 (siRNA molecule composed of a nucleotidesequence described in any of SEQ ID NOs: 724 to 809 and 974 to 1063, anda strand complementary thereto),

(18) a method for inducing apoptosis of target cells comprising a stepof administering (contacting) any of the apoptosis-inducing agents tothe cells,

(19) a method for treating cancer comprising a step of administering theapoptosis-inducing agent or anticancer agent to an individual (e.g., acancer patient),

(20) use of a compound which inhibits chromosome stabilization (forexample, a compound which inhibits expression of any of the genesdescribed in (4) above or inhibits the function of a protein encoded bysaid genes) to produce an apoptosis-inducing agent, and(21) use of the apoptosis-inducing agent to produce an anticancer agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the names of genes used in Examples, accession numbers,siRNA sequences, SEQ ID NOs, inhibition of gene expression in HeLacells, MTT assay (HeLa cells), results of the TUNEL method, inhibitionof gene expression in TIG3 cells, and MTT assay (TIG3 cells).

The column entitled “Inhibition of gene expression in HeLa cells”indicates the results of respectively introducing siRNA for each geneinto HeLa cells, and quantifying expression of each mRNA by Taqman PCR48 hours after introduction.

The column entitled “MTT assay (HeLa cells)” indicates the results ofrespectively introducing siRNA for each gene into HeLa cells, andinvestigating the cell survival rates by an MTT assay 4 days afterintroduction.

The column entitled “TUNEL method” shows YES if staining has beenobserved, i.e., when it was apoptosis-positive.

The column entitled “Inhibition of gene expression in TIG3 cells”indicates the results of respectively introducing siRNA for each geneinto TIG3 cells and quantifying expression of mRNA 72 hours later byTaqman PCR. ND stands for “not detectable”.

The column entitled “MTT assay (TIG3 cells)” indicates the results ofrespectively introducing siRNA for each gene into TIG3 cells andinvestigating the cell survival rates 4 days later by an MTT assay.

The genes were grouped according to their respective functions.

FIG. 2 is a continuation of FIG. 1.

FIG. 3 is a continuation of FIG. 2.

FIG. 4 is a continuation of FIG. 3.

FIG. 5 shows photographs indicating induction of apoptosis by inhibitionof mRNA expression of each gene in HeLa cells. The photographs show theresults of respectively introducing siRNA for each gene into HeLa cellsand examining induction of apoptosis in the HeLa cells 48 hours afterintroduction using the TUNEL method. The green color on the left side ofeach panel (black-and-white photographs are shown) indicates apoptoticnuclei, and the right side indicates nuclei of cells present in thefield of view.

FIG. 6 is a continuation of FIG. 5.

FIG. 7 is a continuation of FIG. 6.

FIG. 8 is a continuation of FIG. 7.

FIG. 9 is a continuation of FIG. 8.

FIG. 10 shows photographs indicating the results of immunostaining theregions in which single-strand DNA is exposed in chromosomal DNA usinganti-ssDNA antibody. Three photographs are shown for each gene. Startingfrom the left, an anti-ssDNA image, nuclear staining image, andsuperimposed image, are shown.

FIG. 11 is a continuation of FIG. 10.

FIG. 12 is a continuation of FIG. 11.

FIG. 13 is a continuation of FIG. 12.

FIG. 14 is a continuation of FIG. 13.

FIG. 15 is a continuation of FIG. 14.

FIG. 16 is a continuation of FIG. 15.

FIG. 17 is a continuation of FIG. 16.

FIG. 18 is a continuation of FIG. 17.

FIG. 19 is a continuation of FIG. 18.

FIG. 20 is a continuation of FIG. 19.

FIG. 21 is a continuation of FIG. 20.

FIG. 22 is a continuation of FIG. 21.

FIG. 23 is a continuation of FIG. 22.

FIG. 24 is a continuation of FIG. 23.

FIG. 25 is a continuation of FIG. 24.

FIG. 26 is a continuation of FIG. 25.

FIG. 27 is a continuation of FIG. 26.

FIG. 28 shows the names of genes used in Examples, accession numbers,other accession numbers, siRNA sequences, SEQ ID NOs, inhibition of geneexpression in HeLa cells, inhibition of proliferation in HeLa cells,inhibition of gene expression in TIG3 cells, and inhibition ofproliferation in TIG3 cells.

The column entitled “Inhibition of gene expression in 40 nM HeLa cells”indicates the results of respectively introducing an siRNA sequence foreach gene into HeLa cells, and quantifying the expression of each mRNAby Taqman PCR 48 hours after introduction.

The column entitled “Inhibition of proliferation in 40 nM HeLa cells”indicates the results of respectively introducing an siRNA sequence foreach gene into HeLa cells, and investigating the cell survival rates byan MTT assay 4 days after introduction.

The column entitled “Inhibition of gene expression in 40 nM TIG3 cells”indicates the results of respectively introducing siRNA for each geneinto TIG3 cells, and quantifying the expression of mRNA 72 hours laterby Taqman PCR. The symbol “**” indicates “not determined”.

The column entitled “Inhibition of proliferation in 40 nM TIG3 cells”indicates the results of respectively introducing an siRNA sequence foreach gene into TIG3 cells, and investigating the cell survival rates byan MTT assay 4 days after introduction.

The genes were grouped according to their respective functions.

FIG. 29 is a continuation of FIG. 28.

FIG. 30 is a continuation of FIG. 29.

FIG. 31 shows alternative names for KNTC2 (NDC80) gene, accessionnumber, mRNA registrations, siRNA IDs, siRNA sequences, SEQ ID NOs, mRNAexpression in HeLa cells, inhibition of proliferation in HeLa cells,apoptosis in HeLa cells, mRNA expression in HDF cells, inhibition ofproliferation in HDF cells, and apoptosis in HDF cells.

The column entitled “mRNA expression” in HeLa cells indicates theresults of respectively introducing an siRNA sequence for KNTC2 (NDC80)gene into HeLa cells, and quantifying expression of each mRNA by TaqmanPCR 48 hours after introduction.

The column entitled “Inhibition of proliferation” in HeLa cellsindicates the results of respectively introducing an siRNA sequence forKNTC2 (NDC80) gene into HeLa cells, and investigating the cell survivalrates by an MTT assay 4 days after introduction.

The column entitled “Apoptosis” in HeLa cells shows YES if staining wasobserved, i.e., when it was apoptosis-positive.

The column entitled “mRNA expression” in HDF cells indicates the resultsof respectively introducing an siRNA sequence for KNTC2 (NDC80) geneinto HDF cells, and quantifying the expression of each mRNA by TaqmanPCR 48 hours after introduction.

The column entitled “Inhibition of proliferation” in HDF cells indicatesthe results of respectively introducing an siRNA sequence for KNTC2(NDC80) gene into HDF cells, and investigating the cell survival ratesby MTT assay 4 days after introduction.

The column entitled “Apoptosis” in HDF cells shows YES if staining wasobserved, i.e., when it was apoptosis-positive.

FIG. 32 shows the names of genes used in Examples, siRNA IDs, siRNAsequences, SEQ ID NOs, mRNA expression in HeLa cells, inhibition ofproliferation in HeLa cells, apoptosis in HeLa cells, mRNA expression inHDF cells, inhibition of proliferation in HDF cells, and apoptosis inHDF cells.

The column entitled “Expression” of mRNA in HeLa cells indicates theresults of respectively introducing an siRNA sequence for each gene intoHeLa cells, and quantifying expression of each mRNA by Taqman PCR 48hours after introduction.

The column entitled inhibition of “Proliferation” in HeLa cellsindicates the results of respectively introducing an siRNA sequence foreach gene into HeLa cells, and investigating the cell survival rates byan MTT assay 4 days after introduction.

The column entitled “Apoptosis” in HeLa cells shows “+” if staining wasobserved, i.e., when it was apoptosis-positive.

The column entitled “Expression” of mRNA in HDF cells indicates theresults of respectively introducing an siRNA sequence for each gene intoHDF cells, and quantifying expression of each mRNA by Taqman PCR 48hours after introduction.

The column entitled inhibition of “Proliferation” in HDF cells indicatesthe results of respectively introducing an siRNA sequence for each geneinto HDF cells, and investigating the cell survival rates by MTT assay 4days after introduction.

The column entitled “Apoptosis” in HDF cells shows “+” if staining wasobserved, i.e., when it was apoptosis-positive, and shows “−” when itwas apoptosis-negative.

FIG. 33 shows photographs indicating induction of apoptosis byinhibiting the mRNA expression of Pif1, Mms4, Topoisomerase IIIa, Mus81,SIRT1 (Sirtuin), Esp1, MPG, Poll, Polm, and EndoV gene in HeLa cells andTIG3 cells. The photographs show the results of respectively introducingsiRNA for each gene into HeLa cells and TIG3 cells, and examining theinduction of apoptosis in HeLa cells 48 hours after introduction and inTIG3 cells 72 hours after introduction using the TUNEL method.

FIG. 34 shows photographs continuing from FIG. 33.

FIG. 35 shows photographs indicating induction of apoptosis byinhibiting the mRNA expression of KNTC2 (NDC80) gene in HeLa cells andTIG3 cells. The photographs show the results of respectively introducingsiRNA for KNTC2 (NDC80) gene into HeLa cells and TIG3 cells, andexamining the induction of apoptosis in HeLa cells 48 hours afterintroduction and in TIG3 cells 72 hours after introduction using theTUNEL method. The photographs on the left side depict apoptotic nuclei.The photographs on the right depict nuclei of cells present in the fieldof view.

FIG. 36 shows photographs indicating the results of immunostaining theregions in which single-strand DNA is exposed in chromosomal DNA usingan anti-ssDNA antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors found that inhibition of chromosome stabilizationinduces cancer cell (tumor cells)-specific apoptosis.

First, the present invention provides cancer cell-specific (anti-cancercell) apoptosis-inducing agents comprising a compound that inhibitschromosome stabilization.

The apoptosis-inducing agents of the present invention are characterizedin that they have an action to selectively induce apoptosis in cancercells. In the present invention, “cancer cell-specific” means that theagent substantially demonstrates an apoptosis-inducing action in cancercells without demonstrating a substantial apoptosis-inducing action innormal cells. Preferably, it means that the agent has anapoptosis-inducing action against cancer cells without showing anapoptosis-inducing action against normal cells.

The term “apoptosis” generally refers to cell death actively induced bythe cell itself due to a physiological condition. Morphological featuresof apoptosis include, for example, chromosome condensation in the cellnucleus, nuclear fragmentation, loss of microvilli on the cell surface,and cytoplasmic shrinkage. Thus, as used herein, the term“apoptosis-inducing action” refers to, for example, the action ofinducing in cells any of the above-described morphological features ofapoptosis, but is not limited to those described above. One skilled inthe art can appropriately assess whether apoptosis induction is takingplace in cells or not.

The cancer cell-specific apoptosis-inducing agents of the presentinvention are considered to be, for example, anticancer agents(carcinostatics) having an apoptosis-inducing action as a mechanism offunction. Since the apoptosis-inducing agents of the present inventionspecifically induce apoptosis in cancer cells but do not induceapoptosis in normal cells, they are expected to be safe anticanceragents having few adverse side effects.

The “anticancer agent” as used herein, may also be referred to as a“carcinostatic agent”. The “anticancer agent” may also be expressed asan “antitumor agent”, “antitumor pharmaceutical”, “antitumorpharmaceutical composition”, etc.

In the present invention, “inhibition of chromosome stabilization”indicates, for example, reaching a state in which unrepaired damageremaining in chromosomal DNA has accumulated, and more specifically, astate in which regions with exposed single strand chromosomal DNA haveaccumulated, or a state in which a large number of breaks indouble-strand DNA have appeared; however, “inhibition of chromosomestabilization” is not necessarily limited to these states.

In the present invention, “chromosome stabilization” is maintained, forexample, by the following functions in cells. Thus, inhibition of thefollowing functions inhibits chromosome stabilization

(a) genes associated with human chromosomal instability disorders,

(b) chromosomal DNA replication reaction including initiation ofchromosomal DNA replication and progression of replication fork,

(c) DNA damage checkpoints,

(d) sister chromatid agglutination and separation,

(e) base excision repair,

(f) mismatch excision repair,

(g) nucleotide excision repair,

(h) homologous recombination repair,

(i) non-homologous end-joining repair (non-homologous recombinationrepair),

(j) double-strand DNA break repair,

(k) DNA post-replication repair (DNA damage tolerance),

(l) DNA crosslink damage repair,

(m) DNA-protein crosslink damage repair,

(n) DNA polymerase,

(o) nuclease,

(p) nucleotide cleansing,

(q) chromatin structure maintenance, and

(r) telomere structure maintenance.

In a preferred embodiment of the present invention, inhibition ofchromosome stabilization includes inhibition of any of theaforementioned functions (a) to (r).

Namely, a preferred embodiment of the present invention relates tocancer cell-specific apoptosis-inducing agents containing a compoundwhich inhibits any of the aforementioned functions (a) to (r).

In the present invention, in order to inhibit any of the aforementionedfunctions (a) to (r), for example, the expression of a gene associatedwith the function (which may also be referred to as a “chromosomestabilization-associated gene” in the present specification) may beinhibited, or the function (activity) of a protein encoded by the genemay be inhibited.

Although examples of genes associated with each of the aforementionedfunctions are provided below, there are no particular limitations solong as they are genes associated with each of the aforementionedfunctions.

(a) Genes Associated with Human Chromosomal Instability Disorders

Examples of human chromosomal instability disorders include xerodermapigmentosum, Cockayne syndrome, Nijmegen breakage syndrome, ataxiatelangiectasia, Fanconi's anemia, and progeria Genes associated withthese diseases are described below.

Xeroderma pigmentosum: (a1) XPB, (a2) XPD, (a3) XPG, (a4) XPF, (a5) XPC,(a6) RAD23B, (a7) CETN2, (a8) RAD23A, (a9) ERCC1

Cockayne syndrome: (a10) CSA, (a11) CSB, (a12) XAB

Nijmegen breakage syndrome: (a13) NBS1

Ataxia telangiectasia: (a14) ATM

Fanconi's anemia: (a15) FANCA, (a16) FANCC, (a17) FANCD2, (a18) FANCE,(a19) FANCF, (a20) FANCG

Progeria: (a21) WRN, (a22) BLM, (a23) RTS

(b) Chromosomal DNA Replication Reaction Including Initiation ofChromosomal DNA Replication and Progression of Replication Fork

(b1) Mcm10, (b2) Orc1, (b3) Orc3, (b4) Cdc6, (b5) Cdt1, (b6) Geminin,(b7) Mcm3, (b8) Mcm4, (b9) Mcm5, (b10) Mcm6, (b11) Mcm7, (b12) Mcm8,(b113) Cdc7, (b14) Cdc5, (b15) Psf1, (b16) Psf2, (b17) Psf3, (b18)Cdc45, (b19) Pola p180, (b20) Pola p70, (b21) Pola Spp1 (Prim2a), (b22)RPA70, (b23) RPA34, (b24) PCNA, (b25) Elg1, (b26) Ligase1, (b27) PolePol2, (b28) Pole Dpb3, (b29) Topoisomerase I, (b30) TDP1, (b31) Orc2,(b32) Orc4, (b33) Orc5, (b34) Orc6, (b35) Mcm2, (b36) Dbf4, (b37)TopBP1, (b38) Sld5, (b39) Pola Spp2, (b40) RFC1, (b41) RFC2, (b42) RFC3,(b43) RFC4, (b44) RFC5, (b45) Pif1, (b46) Pold p50, (b47) Pole Dpb2,(b48) Topoisomerase Iia, (b49) Topoisomerase Iib, (b50) RPAI4, (b51)FEN1, (b52) DNA2, (b53) Pold p125, (b54) Pold p68, (b55) Pold p12, (b56)Pole Dpb4(c) DNA Damage Checkpoints(c1) ATR, (c2) Chk1, (c3) NBS1, (c4) Hus1, (c5) Rad1, (c11) Mad2, (c12)BubR1, (c12) ATM, (c13) Rad50, (c14) Mre11, (c15) Mdc1, (c16) 53BP1,(c17) Rad17, (c22) BubR1, (c23) ATRIP, (c24) Chk2, (c25) H2AX, (c26)RFC1, (c27) RFC2, (c28) RFC3, (c29) RFC4, (c30) RFC5, (c31) ATM, (c32)BRCA1, (c33) Chk1, (c34) Chk2, (c35) 14-3-3eta, (c36) 14-3-3sigma, (c37)cdc25A, (c38) cdc25c, (c39) wee1, (c40) ATR, (c41) ATRIP, (c42) Rad17,(c43) RFC2, (c44) RFC3, (c45) RFC4, (c46) RFC5, (c47) HUS1, (c48) Rad1,(c49) Rad9, (c50) P53, (c51) Rad50, (c52) Mre11, (c53) NBS1, (c54)TopBP1, (c55) 53BP1, (c56) H2AX(d) Sister Chromatid Agglutination and Separation(d1) Ctf18, (d2) Scc1, (d3) Scc3, (d4) Dcc1, (d5) Trf4-1, (d6) Trf4-2,(d7) Smc1, (d8) Smc3, (d9) Pds1 (Securin), (d10) Mad-2, (d11) BubR1,(d12) Esp1(e) Base Excision Repair(e1) UNG (e2) MBD4, (e3) TDG, (e4) NTH1, (e5) NEIL2, (e6) NEIL3, (e7)APE2, (e8) PARP1, (e9) PNK, (e10) Polb, (e11) OGG1, (e12) APE1, (e13)XRCC1, (e14) Ligase3, (e15) SMUG1, (e16) TDG, (e17) MYH, (e18) MPG,(e19) NEIL1, (e20) ADPRT, (e21) ADPRTL2, (e22) MGMT, (e23) ABH1, (e24)ABH2, (e25) ABH3(f) Mismatch Excision Repair(f1) MSH2, (f2) PMS1, (f3) PMS2, (f4) MLH3, (f5) Exonuclease1, (f6)MSH3, (f7) MSH6, (f8) MSH5, (f9) MLH1, (f10) MSH4, (f11) PMS2L3, (f12)Trex1, (f13) Trex2, (f14) PMS2L4(g) Nucleotide Excision Repair(g1) XPC, (g2) Rad23A, (g3) Rad23B, (g4) CSA, (g5) CSB, (g6) XPG, (g7)XPF, (g8) DDB1, (g9) DDB2, (g10) XAB2, (g11) XPB, (g12) ERCC1, (g13)XPD, (g14) XPA, (g15) DDB2, (g16) Mms19, (g17) CETN2, (g18) RPA70, (g19)RPA34, (g20) RPAI4, (g21) GTF2H1, (g22) GTF2H2, (g23) GTF2H3, (g24)GTF2H4, (g25) CDK7, (g26) CCNH, (g27) MNAT1, (g28) LigaseI, (g29) CSA,(g30) CSB(h) Homologous Recombination Repair(h1) Rad51, (h2) Rad51L1, (h3) Rad51C, (h4) Rad51L3, (h5) DMC1, (h6)XRCC2, (h7) XRCC3, (h8) Rad52, (h9) Rad54L, (h10) Rad54B, (h11) BRCA1,(h12) BRCA2, (h13) Rad50, (h14) Mre11, (h15) NBS1, (h16)TopoisomeraseIIIa, (h17) TopoisomeraseIIIb, (h18) WHIP, (h19) WRN, (h20)BLM, (h21) RecQ1, (h22) RecQ5(i) Non-Homologous End-Joining Repair (Non-Homologous RecombinationRepair)(i1) Ku70, (i2) Ku80, (i3) DNA-pk, (i4) Ligase4, (i5) XRCC4, (i6)Artemis, (i7) WRNj) Double-Strand DNA Break Repair(j1) Rad51, (j2) Rad51D, (j3) Xrcc2, (j4) Rad54, (j5) BRCA1, (j6) Ku80,(j7) XRCC4, (j8) Rad52, (j9) Rad51C, (j10) Dmc1, (j11) Rad54B, (j12)DNA-pk, (j13) Ku70, (j14) Ligase4, (j15) Rad51B, (j16) XRCC3, (j17)BRCA2, (j18) Artemis(k) DNA Post-Replication Repair (DNA Damage Tolerance)(k1) Rad6A, (k2) Rad6B, (k3) Rad18, (k4) Ubc13, (k5) FBH1(l) DNA Crosslink Damage Repair(l1) FANCA, (l2) FANCC, (l3) FANCD2, (l4) FANCE, (l5) FANCF, (l6) FANCG(m) DNA-Protein Crosslink Damage Repair(m1) TDP1(n) DNA Polymerase(n1) Poli, (n2) Polh, (n3) Polq, (n4) Polk, (n5) Polz (REV3), (n6) Poll,(n7) Polm, (n8) Rev1, (n9) Polb, (n10) Polg, (n11) Pold p50, (n12) PolePol2, (n13) REV7, (n14) Poln, (n15) Pola P180, (n16) Pola p70, (n17)Pola Spp1, (n18) Pola Spp2, (n19) Pold p68, (n20) Pold p12, (n21) PoleDpb2, (n22) Pole Dpb3, (n23) Pole Dpb4(o) Nuclease(o1) FEN1, (o2) TREX1, (o3) TREX2, (o4) Exonuclease1, (o5) SPO11, (o6)ENDO V, (o7) APE1, (o8) APE2, (o9) Mre11, (o10) Artemis(p) Nucleotide Cleansing(p1) MTH1, (p2) DUT, (p3) p53R2(q) Chromatin Structure Maintenance(q1) H2AX, (q2) Sir2, (q3) SIRT1 (Sirtuin)(r) Telomere Structure Maintenance(r1) Tin2, (r2) Sir2, (r3) hTert, (r4) TRF1, (r5) TRF2, (r6) Tankyrase,(r7) Pot1, (r8) Rap1, (r9) Pif1

Preferred examples of genes associated with each of the aforementionedfunctions (a) to (r) include the genes described in Examples below. Morespecifically, examples of such genes are as follows:

APE2, ATR, BRCA1, Chk1, Cdc5, Cdc6, Cdc7, Cdc45, Cdt1, CSA, CSB, Ctf18,DDB1, DDB2, DNA2, DUT, Elg1, EndoV, Esp1, Exonuclease1, FBH1, FEN1,Geminin, Hus1, KNTC2 (NDC80), Ku80, Ligase1, Mad2, MBD4, Mcm3, Mcm4,Mcm5, Mcm6, Mcm7, Mcm8, Mcm10, MGMT, MLH3, Mms4, MPG, MSH2, Mus81, NBS1,NEIL2, NEIL3, NTH1, Orc1, Orc3, PARP1, PCNA, Pif1, PMS1, PMS2, PNK, Polap180, Pola p70, Pola Spp1 (Prim2a), Polb, Pold p125, Pole Dpb3, PoleDpb4, Pole Pol2, Poli, Poll, Polm, Psf1, Psf2, Psf3, Rad1, Rad18,Rad23A, Rad23B, Rad51, Rad51D, Rad54, Rad6A, RPA34, RPA70, Scc1, Scc3,Sir2, SIRT1 (Sirtuin), TDG, TDP1, TIMELESS, Tin2, Topoisomerase I,Topoisomerase IIIa, Topoisomerase IIIb, Ubc13, UNG, XAB2, XPC, XPF, XPG,Xrcc2, and XRCC4.

A preferred embodiment of the present invention provides a cancercell-specific apoptosis-inducing agent comprising as an activeingredient a compound which inhibits the expression of a chromosomestabilization-associated gene (for example, any of the aforementionedgenes), or inhibits the function of a protein encoded by the gene.

Since the gene names described in the present specification are nameswhich are widely and generally known, those skilled in the art are ableto suitably acquire data on the nucleotide sequences of said genes froma public reference database or gene database (e.g., GenBank) based onthe gene name.

Specific examples of the nucleotide sequences of the aforementionedgenes of the present invention and amino acid sequences of proteinsencoded by the genes are listed in the Sequence Listing. NCBI accessionnumbers by which sequence data on the genes can be acquired, and therelationships between the nucleotide sequences of genes acquired usingsaid numbers and SEQ ID NOs, are shown in Tables 1 to 16. In addition,examples of amino acid sequences of proteins encoded by theaforementioned genes of the present invention are also shown in theSequence Listing.

TABLE 1 SEQ ID NO Amino Gene Name Accession No. Nucleotide Sequence AcidSequence Mcm10 NM_182751 1 638 NM_018518 2 AB042719 3 AL136840 4AK055695 5 BC009108 6 BC004876 7 AF119869 8 Orc1 NM_004153 9 639 U4341610 U40152 11 BC011539 12 Orc3 NM_181837 13 640 NM_012381 14 BC035494 15AF125507 16 AF135044 17 AL080116 18 AF093535 19 BC047689 20 U50950 21AK094135 22 Cdc6 NM_001254 23 641 AF022109 24 BC025232 25 U77949 26 Cdt1NM_030928 27 642 BC008676 28 AF321125 29 AB053172 30 BC000137 31BC008860 32 BC009410 33 BC049205 34 BC021126 35 AF070552 36 BC014202 37Geminin NM_015895 38 643 BC005389 39 BC005185 40 AF067855 41 AK021685 42Mcm3 BC003509 43 NM_002388 44 644 BC001626 45 AY032603 46 X62153 47D38073 48 U41843 49

TABLE 2 Mcm4 NM_005914 50 645 XM_030274 51 X74794 52 NM_182746 53BC031061 54 AK022899 55 Mcm5 NM_006739 56 646 X74795 57 BC003656 58BC000142 59 D83986 60 AK130620 61 AK122853 62 Mcm6 NM_005915 72 647BC020268 73 D84557 74 BC032374 75 U46838 76 BC008774 77 Mcm7 NM_00591665 648 BC013375 63 D55716 70 AK096959 71 X74796 68 NM_182776 64 D2848066 AK055379 67 AY007130 69 BC009398 78 AF279900 79 Mcm8 NM_032485 80 649AJ439063 81 BC008830 82 AK027644 83 NM_182802 84 AY158211 85 BC005170 86

TABLE 3 Cdc7 NM_003503 87 650 AF015592 88 AB003698 89 AF005209 90 Cdc5BC001568 91 NM_001253 92 651 U86753 93 AK128737 94 AB007892 95 D85423 96Psf1 NM_021067 97 652 D80008 98 BC012542 99 Psf2 BC010164 100 NM_016095101 653 AF151880 102 AF125098 103 AK001275 104 AF201939 105 BC022839 106BC003186 107 BC062444 108 AK091519 109 Psf3 NM_022770 110 654 BC014437111 BC005879 112 AK127454 113 AK023974 114 AL137379 115 Cdc45 BC005879112 NM_003504 116 655 BC006232 117 BT006792 118 BC010022 119 AF081535120 AF053074 121 AY358971 122 AF062495 123 AJ223728 124 Pola p180NM_016937 125 656 X06745 126 BX648513 127 Pola p70 L24559 128 BC002990129 NM_002689 130 657 BC001347 131 BC018813 132 BC018814 133 AK025315134 AK094569 135 Pola Spp1(Prim2a) NM_000947 136 658 X74331 137 BC017833138

TABLE 4 RPA70 BC018126 139 NM_002945 140 659 M63488 141 RPA34 NM_002946142 660 BC021257 143 BC012157 144 BC001630 145 J05249 146 PCNA NM_002592147 661 NM_182649 148 BC000491 149 M15796 150 Elg1 AJ314648 151NM_024857 152 662 AL832103 153 AK022797 154 BC015051 155 FEN1 NM_004111156 663 BC000323 157 X76771 158 L37374 159 XM_209325 160 DNA2 D42046 161XM_166103 162 664 BC063664 163 BC053574 164 BC041115 165 BC028188 166BC017003 167 Ligase1 NM_000234 168 665 M36067 169 Pold p125 NM_002691170 666 M80397 171 BC008800 172 M81735 173 Pole Pol2 NM_006231 174 667L09561 175 U49356 176 S60080 177 BX647647 178 BC007599 179 BC021559 180AK093003 181 AK025087 182 BC011376 183 AL080203 184 AK128248 185NM_012332 186 AF132950 187

TABLE 5 Pole Dpb3 NM_017443 188 668 AK074762 189 BC004170 190 BC003166191 AK074629 192 AF226077 193 AK074782 194 AK096050 195 AK092840 196Pole Dpb4 AF261688 197 BC031331 198 NM_019896 199 669 AY034104 200Topoisomerase I NM_003286 201 670 J03250 202 U07806 203 U07804 204X16479 205 TDP1 BC015474 206 NM_018319 207 671 AK001952 208 AF182002 209BX161451 210 AK093235 211 BC006083 212 AL832288 213 AF182003 214AK023514 215 Ctf18 BC018184 216 NM_022092 217 672 BC006278 218 BC006437219 AK024476 220 AK128869 221 Scc1 BC050381 222 NM_006265 223 673 D38551224 X98294 225 AK098521 226 AK097915 227 BC001229 228 AK125620 229

TABLE 6 Scc3 NM_005862 230 674 Z75330 231 BC017735 232 BC040708 233AF070586 234 BC001765 235 NM_006603 236 BX641003 237 AK098737 238 Z75331239 BX641002 240 BX640970 241 AL831939 242 AK124202 243 NM_012447 244AJ007798 245 BC047490 246 BC028684 247 ATR NM_001184 248 675 Y09077 249U76308 250 U49844 251 Chk1 BC017575 252 NM_001274 253 676 AF016582 254BC004202 255 AF032874 256 NBS1 NM_002485 257 677 AF051334 258 AF058696259 BX640816 260 BC040519 261 BC005293 262 BC016762 263 AK001017 264Hus1 NM_004507 265 678 BC007013 266 AF110393 267 AF076844 268 Y16893 269AJ227901 270 AK097182 271

TABLE 7 Rad1 BC037857 272 BC009804 273 NM_133377 274 NM_002853 275 679BC006837 276 AK002112 277 AF074717 278 AF076841 279 AF030933 280AF084512 281 AF058392 282 AF011905 283 AJ004974 284 BT006908 285AF073524 286 NM_133282 287 AF090170 288 AF084513 289 AF058393 290AJ004975 291 Topoisomerase IIIb NM_003935 292 680 AF053082 293 AF017146294 AF125216 295 BC002432 296 AL833505 297 AK096695 298 AF070585 299XM_066339 300 BC051748 301 NM_004618 302 U43431 303 Rad6A BC010175 304NM_003336 305 681 M74524 306 NM_181777 307 BC042021 308 NM_181762 309BC005979 310 BC008404 311 BC008470 312 NM_003337 313 BT007071 314 X53251315 M74525 316 Rad18 NM_020165 317 682 AF169796 318 AK023075 319AB035274 320 BC001302 321 AY004333 322

TABLE 8 Ubc13 BC000396 323 BC003365 324 NM_003348 325 683 D83004 326BT006873 327 XM_372257 328 AK098233 329 FBH1 NM_178150 330 NM_032807 331684 AF380349 332 AF456237 333 AK095343 334 AF454502 335 BC020266 336BC032674 337 AK122753 338 AK027496 339 AK027381 340 BC006430 341BC012762 342 AL133069 343 AL832251 344 Mad2 NM_002358 345 685 BC000356346 BC005945 347 U31278 348 AJ000186 349 U65410 350 NG_002592 351AF394735 352 XM_374193 353 XPC BC016620 354 NM_004628 355 686 D21089 356X65024 357 Rad23A BC014026 358 NM_005053 359 687 D21235 360 M77024 361L37720 362 BC020973 363 NM_002874 364 AY313777 365 AK125226 366 D21090367

TABLE 9 Rad23B NM_002874 364 688 D21090 367 BC020973 363 AK125226 366AY313777 365 XM_067249 368 BC014026 358 NM_005053 359 D21235 360AK122683 369 CSA NM_000082 370 689 U28413 371 AK056931 372 BC009793 373CSB NM_000124 374 690 L04791 375 AK130100 376 XPG X69978 377 NM_000123378 691 BC031522 379 AF462447 380 BX647399 381 L20046 382 D16305 383 XPFL77890 384 NM_005236 385 692 U64315 386 BC020741 387 DDB1 NM_001923 388693 U32986 389 BC050530 390 BC011686 391 BC051764 392 HSU18299 393AJ2955 394 L40326 395 BC021044 396 BC032080 397 AL831958 398 DDB2NM_000107 399 694 U18300 400 BC000093 401 BT007139 402 BC001160 403BC050455 404 AK091640 405

TABLE 10 XAB2 NM_020196 406 695 AF226051 407 BC007208 408 AF258567 409AB026111 410 AB033003 411 BC008778 412 AK025858 413 AK074035 414 UNGBC050634 415 NM_003362 416 696 BC015205 417 X15653 418 NM_080911 419Y09008 420 MBD4 NM_003925 421 697 AF072250 422 AF114784 423 AF532602 424BC034463 425 BC011752 426 U56428 427 U56254 428 TDG BC037557 429NM_003211 430 698 U51166 431 BC019925 432 BC010945 433 NTH1 NM_002528434 699 U79718 435 AB001575 436 U81285 437 BC000391 438 BC003014 439Y09687 440 NEIL2 BC013964 441 BC013952 442 NM_145043 443 700 AK056206444 AB079070 445 AK097389 446 BX537529 447 BC045822 448 NEIL3 NM_018248449 701 AK001720 450 AB079071 451 BC025954 452 APE2 BC002959 453NM_014481 454 702 AJ011311 455 AB021260 456 AB049211 457 AF119046 458

TABLE 11 PARP1 NM_001618 459 703 M32721 460 M18112 461 J03473 462BC037545 463 NG_002655 464 M17081 465 BC018620 466 BC021045 467 BC014206468 BC008660 469 AK125650 470 AF401218 471 AJ236912 472 AJ236876 473AK001980 474 NM_005484 475 AF085734 476 PNK BC033822 477 NM_007254 478704 AF125807 479 AF126486 480 AF120499 481 BC002519 482 BC009339 483BC013034 484 Polb NM_002690 485 705 D29013 486 L11607 487 M13140 488MSH2 NM_000251 489 706 BC021566 490 L47581 491 U04045 492 L47577 493L47574 494 L47582 495 L47583 496 U03911 497 L47579 498 L47578 499BX649122 500 L47580 501 L47576 502 L47575 503 BC001122 504 BC012599 505PMS1 NM_000534 506 707 U13695 507 BC036376 508 BC008410 509 BT006947 510

TABLE 12 PMS2 NM_000535 511 708 U14658 512 BC031832 513 BC008400 514XM_208368 515 AB116525 516 MLH3 NM_014381 517 709 AF195657 518 AB039667519 Exonuclease1 NM_003686 520 AF091740 521 AF042282 522 NM_006027 523710 BC007491 524 NM_130398 525 AF060479 526 AF084974 527 AL080139 528Poli AF140501 529 NM_007195 530 711 BC032662 531 AF245438 532 AL136670533 BC032617 534 BX649100 535 AK093688 536 Rad51 NM_002875 537 712D14134 538 D13804 539 NM_133487 540 Rad51D NM_002878 541 713 Y15572 542BC014422 543 BX647297 544 AB013341 545 NM_133627 546 AB016223 547AF034956 548 BC002723 549 NM_133628 550 AL117459 551 NM_133630 552AB016224 553 NM_133629 554 AB016225 555 AK097811 556 AB020412 557AB018363 558 AB018360 559 AB018362 560 AB018361 561

TABLE 13 Xrcc2 BC042137 562 NM_005431 563 714 AF035587 564 Y08837 565Rad54 NM_003579 566 715 X97795 567 BRCA1 NM_007295 568 716 NM_007296 569NM_007294 570 NM_007306 571 NM_007302 572 NM_007297 573 U14680 574AF005068 575 NM_007301 576 NM_007300 577 NM_007299 578 Ku80 NM_021141579 717 M30938 580 BC019027 581 J04977 582 X57500 583 XRCC4 NM_022550584 NM_003401 585 718 U40622 586 NM_022406 587 BC016314 588 AB017445 589BC005259 590 BT007216 591 BC010655 592 Tin2 NM_012461 593 719 AF195512594 BC019343 595 BC005030 596 AK023166 597 BX161478 598 Sir2 NM_012237599 720 BC003012 600 BC003547 601 AK025876 602 AF095714 603 AF083107 604NM_030593 605 AJ505014 606 AK054642 607 AF160214 608 AF131800 609AK092940 610

TABLE 14 MGMT NM_002412 611 721 X54228 612 M60761 613 BC000824 614M29971 615 BT006714 616 M31767 617 DUT NM_001948 618 722 AB049113 619BC033645 620 U62891 621 U31930 622 L11877 623 M89913 624 AK000629 625U90223 626 NM_182746 53 BC031061 54 AK022899 55 TIMELESS BC050557 627BC031514 628 AB015597 629 AF098162 630 NM_003920 631 723 BC039842 632AK022702 633 BX640990 634 AK000721 635 AY207390 636 AY207391 637

TABLE 15 SEQ ID NO Nucleotide Amino Gene Name Accession No. SequenceAcid Sequence Pif1 AF108138.1 810 909 BC033254.1 811 AK026345.1 812 910NM_025049.1 813 911 BC018978.2 814 Mms4 NM_152463.1 815 912 AK021607.1816 BC016470.2 817 913 AK055926.1 818 914 TopoisomeraseIIIa NM_004618.2819 915 BC051748.1 820 916 AK126869.1 821 U43431.1 822 917 Mus81NM_025128 823 918 AK126820.1 824 CR604400.1 825 CR601399.1 826AL353934.1 827 919 AK024665.1 828 920 NM_025128.3 829 921 BC009999.2 830922 AF425646.1 831 923 AK095326.1 832 SIRT1 (Sirtuin) NM_012238.3 833924 BX648554.1 834 AF083106.2 835 925 AF235040.1 836 926 AL136741.1 837AK027686.1 838 BC012499.1 839 927 AK074805.1 840 Esp1 NM_012291 841 928BC047603.1 842 929 AK128350.1 843 AY455930.1 844 930 D79987.1 845 931MPG NM_002434 846 932 M99626.1 847 933 NM_002434.1 848 934 CR619346.1849 CR612592.1 850 CR606356.1 851 CR600098.1 852 CR598824.1 853 L10752.1854 935 M74905.1 855 936 X56528.1 856 937 BC014991.1 857 938 M71215.1858 939 S51033.1 859 940

TABLE 16 Poll NM_013274 860 941 AK128521.1 861 AK127896.1 862 942BC068529.1 863 943 AJ131890.1 864 944 CR619817.1 865 CR615868.1 866NM_013274.2 867 945 AF161019.1 868 946 AK021600.1 869 947 AK022476.1 870948 AF218027.1 871 949 AF283478.1 872 950 BC003548.1 873 951 AK094956.1874 Polm NM_013284 875 952 BC049202.1 876 953 BC062590.1 877 954BC026306.1 878 955 AJ131891.2 879 956 CR620839.1 880 CR606869.1 881NM_013284.1 882 957 AF176097.1 883 958 AK023002.1 884 959 AK092903.1 885AK092801.1 886 960 BC035685.1 887 EndoV NM_173627 888 961 NM_173627.2889 962 BC045824.1 890 963 BX647411.1 891 AK123689.1 892 964 BC059781.1893 BC064545.1 894 965 CR617882.1 895 CR599326.1 896 AK056045.1 897AK096802.1 898 AK096344.1 899 966 AK092539.1 900 967 BC037889.2 901 968KNTC2 (NDC80) NM_006101 902 969 NM_006101.1 903 970 CR609890.1 904BC010171.2 905 971 BC005239.1 906 BC035617.1 907 972 AF017790.1 908 973

Each of the aforementioned genes may be assigned multiple accessionnumbers even for the same gene due to the presence of polymorphisms inthe nucleotide sequence or the like. These “polymorphisms” are notlimited to single nucleotide polymorphisms (SNPs) including a mutationof a single nucleotide by substitution, deletion, or insertion, and alsoinclude substitutions, deletions, and insertion mutations of severalcontiguous nucleotides. Thus, the nucleotide sequences of theaforementioned genes are not necessarily limited to sequences acquiredaccording to the accession numbers described in Tables 1 to 16, or tothe sequences described in SEQ ID NOs. 1 to 637 and 810 to 908.Similarly, the amino acid sequences of proteins encoded by theaforementioned genes are not particularly limited to the amino acidsequences described in SEQ ID NOs. 638 to 723 and 909 to 973.

The aforementioned proteins of the present invention are not limited tothe amino acid sequences described in SEQ ID NOs. 638 to 723 and 909 to973, and include proteins comprising amino acid sequences in which oneor more of the amino acid residues in said amino acid sequences havebeen added, deleted, substituted, or inserted, and which arefunctionally equivalent to the proteins described in SEQ ID NOs. 638 to723 and 909 to 973.

The chromosome stabilization-associated genes of the present invention(e.g., the aforementioned various genes) are normally of animal origin,more preferably of mammalian origin, and most preferably of humanorigin, but they are not particularly limited thereto.

Namely, the present invention is not limited to apoptosis-inducingagents specific for human cancer cells, and also includesapoptosis-inducing agents for cancer cells of nonhuman animals. Thus,nonhuman-animal homolog (counterpart) genes of the aforementioned genesare included in the genes of the present invention. For example,endogenous genes (e.g., homologs) in other animals corresponding togenes comprising each of the nucleotide sequences described in SEQ IDNOs: 1 to 637 and 810 to 908 are included. Endogenous DNA of otheranimals corresponding to DNA comprising the nucleotide sequencesgenerally has high homology with DNA described in the SEQ ID NOs above.High homology refers to homology of 50% or more, preferably 70% or more,more preferably 80% or more, and even more preferably 90% or more (forexample, 95% or more, or further 96%, 97%, 98%, or 99% or more). Thehomology can be determined by the mBLAST algorithm (Altschul et al.(1990), Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul(1993), Proc. Natl. Acad. Sci. USA 90: 5873-7). In addition, thehomologous DNA is thought to hybridize under stringent conditions withDNA described in the above SEQ ID NOs if it has been isolated from theliving body. Here, “stringent conditions” are, for example, “2×SSC, 0.1%SDS, 50° C.”, “2×SSC, 0.1% SDS, 42° C.”, or “1×SSC, 0.1% SDS, 37° C.”,and more stringent conditions are “2×SSC, 0.1% SDS, 65° C.”, “0.5×SSC,0.1% SDS, 42° C.”, or “0.2×SSC, 0.1% SDS, 65° C.”. Those skilled in theart are able to suitably acquire data (such as sequence data) relatingto endogenous genes corresponding to each of the aforementioned genes ofthe present invention in other animals based on the nucleotide sequencesdescribed in the Sequence Listing.

In addition, the present invention provides compounds which inhibitexpression of chromosome stabilization-associated genes (for example,any of the aforementioned genes).

Preferred examples of compounds of the present invention which inhibitexpression of chromosome stabilization-associated genes (for example,any of the aforementioned genes) include double-strand RNA having anRNAi (RNA interference) effect on said genes. In general, the term“RNAi” refers to a phenomenon where target gene expression is inhibitedby inducing disruption of the target gene mRNA. This disruption iscaused by introducing into cells a double-stranded RNA that comprises,a) a sense RNA comprising a sequence homologous to the target gene mRNAsequence, and b) an antisense RNA comprising a sequence complementary tothe sense RNA.

While details of the RNAi mechanism remains unclear, it is thought thatan enzyme called DICER (a member of the RNase III nuclease family)decomposes double-stranded RNA into small fragments called “smallinterfering RNA” or “siRNA”, when it comes into contact with thedouble-stranded RNA. This siRNA is also included in the double-strandedRNA comprising RNAi activity of the present invention. Furthermore, DNAsthat allow the expression of the double-stranded RNA of the presentinvention are also included in the present invention.

A preferred embodiment of the present invention provides a cancercell-specific apoptosis-inducing agent comprising as an activeingredient a double-strand RNA capable of inhibiting expression of achromosome stabilization-associated gene (for example, any of theaforementioned genes) by an RNAi effect (siRNA), where the doublestranded RNA comprises a structure in which an RNA consisting of anucleotide sequence described in any of SEQ ID NOs: 724 to 809 and 974to 1063, is hybridized with an RNA consisting of a sequencecomplementary to said RNA.

For example, an example of a siRNA of the present invention comprisingthe nucleotide sequence described in SEQ ID NO: 724(5′-ggaaaaucuggccacucucTT-3′) is an RNA molecule having the structureshown below (SEQ ID NOS: 1064 and 1065).

(In the above structure, “|” indicates a hydrogen bond.)

Molecules having a structure in which one end of the above RNA moleculeis closed, such as siRNA having a hairpin structure (shRNA), are alsoincluded in the present invention. Namely, molecules able to form adouble-stranded RNA structure within the molecules are also included inthe present invention.

For example, a molecule such as 5′-ggaaaaucuggccacucuc (xxxx)ngagaguggccagauuuucc-3′ is also included in the present invention (SEQ IDNOS: 1066-1067). (The above “(xxxx)n” represents a polynucleotideconsisting of an arbitrary number of nucleotides or sequences.)

A preferred embodiment of the aforementioned siRNA is a double strandRNA able to inhibit expression of a chromosome stabilization-associatedgene (for example, any of the aforementioned genes) by an RNAi effect(siRNA), comprising a structure in which an RNA consisting of anucleotide sequence described in any of SEQ ID NOs: 724 to 809 and 974to 1063 is hybridized with an RNA consisting of a sequence complementaryto the RNA. However, double-strand RNA, for example, having a structurein which one or more ribonucleotides are added to or deleted from an endof the double-strand RNA, for example, is also included in the presentinvention.

Specifically, the present invention provides DNAs (vectors) that allowthe expression of a double-stranded RNA of the present invention. TheseDNAs (vectors) that allow the expression of a double-stranded RNA of thepresent invention are typically DNAs comprising a structure where a DNAencoding one strand of the double-stranded RNA, and a DNA encoding theother strand of the double-stranded RNA, are operably linked to apromoter. Those skilled in the art can readily prepare anabove-described DNA of the present invention with routinely used geneticengineering techniques. More specifically, expression vectors of thepresent invention can be prepared by appropriately inserting DNAencoding an RNA of the present invention into various known expressionvectors.

Although RNA used for RNAi is not required to be completely identical(homologous) to a chromosome stabilization-associated gene (for example,any of the aforementioned genes) or a partial region of the gene, it ispreferably completely identical (homologous).

The present invention's double-strand RNA having RNAi effects isnormally double-strand RNA comprising sense RNA consisting of a sequencehomologous with an arbitrary contiguous RNA region in the mRNA of achromosome stabilization-associated gene (for example, any of theaforementioned genes), and an antisense RNA consisting of a sequencecomplementary to the sense RNA. The length of the “arbitrary contiguousRNA region” is normally 20 to 30 bases, and preferably 21 to 23 bases.An example includes, but is not necessarily limited to, the length of ansiRNA, having as one of the strands, an RNA described in any of SEQ IDNOs: 724 to 809 and 974 to 1063. However, even in the case of along-strand RNA that does not have RNAi effects as is, the length of thedouble-stranded RNA of the present invention is not limited since thelong-stand is expected to be degraded into siRNA having RNAi effects incells. In addition, long double-strand RNA corresponding to the entirelength or nearly the entire length of the mRNA of a chromosomestabilization-associated gene (for example, any of the aforementionedgenes) can be degraded in advance with, for example, DICER, and theresulting degradation product can be used as an apoptosis-inducing agentof the present invention. This degradation product is expected tocontain a double-strand RNA molecule (siRNA) having RNAi effects. Inthis method, it is not particularly required to select an mRNA regionthat is expected to have an RNAi effect. Namely, it is not necessarilyrequired to accurately define a region on mRNA of a chromosomestabilization-associated gene (for example, any of the aforementionedgenes) that has an RNAi effect. However, the various types of siRNA usedin the Examples described later are more preferred.

In general, double-strand RNA having an overhang of several nucleotideson an end is known to have strong RNAi effects. Double-stranded RNAs ofthe present invention preferably have an overhang of several nucleotideson an end. The length of the nucleotides which form the overhang is notparticularly limited. This overhang may be DNA or RNA. For example, theoverhang preferably has two nucleotides. In the present invention,double-strand RNA having an overhang comprises, for example, TT (twothymines), UU (two uracils), or other nucleotides (most preferablymolecules having double-strand RNA consisting of 19 bases and anoverhang consisting of 2 nucleotides (TT)) can be preferably used.Molecules in which the nucleotides forming the overhang in this mannerare DNA, and sequences homologous to a target mRNA sequence, are alsoincluded in the double-strand RNA of the present invention.

Examples of siRNA molecules of the present invention where thenucleotides of the overhang portion are TT include molecules having TTadded to the 3′ side thereof, such as the molecule indicated below (SEQID NOS: 724 and 1068).

The aforementioned “double-strand RNA having an RNAi effect on achromosome stabilization-associated gene” of the present invention canbe suitably produced by those skilled in the art based on the nucleotidesequence of a chromosome stabilization-associated gene (for example, anyof the aforementioned genes) targeted by said double-strand RNA. Anucleotide sequence of a chromosome stabilization-associated gene (forexample, any of the aforementioned genes) can be easily acquired from apublic gene database as described above. As an example, double-strandRNA of the present invention can be produced based on a nucleotidesequence described in any of SEQ ID NOs: 1 to 637 and 810 to 908.Namely, the selection of an arbitrary contiguous RNA region of mRNA,which is a transcription product of any of the nucleotide sequencesdescribed in SEQ ID NOs: 1 to 637 and 810 to 908, based on thatsequence, and the production of double-strand RNA corresponding to thatregion, can be easily carried out by those skilled in the art. Inaddition, methods for selecting an siRNA sequence having more potentRNAi effects from an mRNA sequence which is a transcript of saidsequences can be suitably carried out by those skilled in the art withreference to, for example, the following documents: Reynold et al.Nature biotechnology 22. 326-330 (2004), Ui-Tei et al. Nucleic AcidsRes. 32. 936-948 (2004), Boese Q, Leake D, Reynolds A, Read S, ScaringeS A, Marshall W S, Khvorova A. Mechanistic insights aid computationalshort interfering RNA design. Methods Enzymol. 2005; 392:73-96., Snove OJr, Nedland M, Fjeldstad S H, Humberset H, Birkeland O R, Grunfeld T,Saetrom P. Designing effective siRNAs with off-target control. BiochemBiophys Res Commun. 2004; 325(3):769-73., Yiu S M, Wong P W, Lam T W,Mui Y C, Kung H F, Lin M, Cheung Y T. Filtering of Ineffective siRNAsand Improved siRNA Design Tool. Bioinformatics. 200515; 21(2):144-51,Chalk A M, Wahlestedt C, Sonnhammer E L. Improved and automatedprediction of effective siRNA. Biochem Biophys Res Commun. 2004;319(1):264-74., Amarzguioui M, Prydz H. An algorithm for selection offunctional siRNA sequences. Biochem Biophys Res Commun. 2004;316(4):1050-8., Sioud M, Leirdal M. Potential design rules and enzymaticsynthesis of siRNAs. Methods Mol Biol. 2004; 252:457-69. In addition, ifone of the strands has been determined (for example, a nucleotidesequence described in any of SEQ ID NOs: 724 to 809 and 974 to 1063),the nucleotide sequence of the other strand (complementary strand) canbe easily determined by those skilled in the art. siRNA can be suitablyproduced by those skilled in the art using a commercially availablenucleic acid synthesizer. To synthesize a desired RNA, custom synthesisservices are also available.

All of the nucleotides in the siRNA of the present invention are notnecessarily required to be ribonucleotides (RNA). Namely, in the presentinvention, one or more of the ribonucleotides which compose the siRNAmay be the corresponding deoxyribonucleotides. This “corresponding”means that the nucleotides have identical base species (adenine,guanine, cytosine, and thymine (uracil)), but the structure of the sugarportion is different. For example, the deoxyribonucleotide correspondingto a ribonucleotide having adenine means a deoxyribonucleotide havingadenine. In addition, the above “more” is not limited to a particularnumber but preferably means a small number around 2 to 5.

It is not essential to have information on the full-length nucleotidesequence of a gene (the target gene) from which the double-stranded RNAof the present invention is derived. It is enough that the arbitrary RNAregion comprising consecutive nucleotides (for example, 20 to 30nucleotides) which is to be selected has been identified. Thus, thedouble-stranded RNA of the present invention can be prepared based onthe nucleotide sequence of a fragment of a gene, such as an ExpressedSequence Tag (EST), whose mRNA sequence has been determined partially,but not completely. The accession numbers and names of EST sequences inthe GenBank database with a high homology to the aforementioned genesare shown below. However, this list includes only a few examples of themany EST sequences. Those skilled in the art can readily obtain sequenceinformation on appropriate EST fragments from public databases.

Mcm10: BQ230201, CK000876, BX324498, BM466246, BI086715, BE561621,BM781578, BE397209, BM781561, BF797760, BE268770, AI33628, BI860489,CA488245, BE018388, BM794417, AI005288, AV759891, BE536223, AI078425,BI023355, CB120985, AA305452, BX324497, AI636632, CB143720, BM465842,BM833978, AI962581, BE206240, BE536858, BQ432059, BX099770, BX346400,BE890219, BU542039, AI750442, BU542210, BQ929138, BG323332, AA312197,BG942019, BM755471, AA091854, BX346399, BG493975, T97047, BU615340,BG777950, BU618352, BG323324Orc1: BM556110, AL558857, AL528479, AL530422, AU125429, BQ229865,BX370588, AL563154, BU552785, BQ055912, CD642483, BE903488, BU854876,BM908744, BF205157, BX372245, BQ230059, BX110476, BM743410, BG821765,AL530421, BI222366, BU198520, BF796650, BM476715, AL552393, AU127396,BM740840, BE781976, AL580583, BG391980, BM852299, CD654548, BQ423782,AW378723, AL582531, AL561250, BU194186, BQ048846, BG822626, AL552501,BU854819, BQ433201, BE782505, BG257286, BX281133, CF140351, BF795920,BG390855, BG831652, BF794915, BF797918, BG325714, AI651655, BM833692,BQ883238, BX474-463, AI038384, AU129034, BP431296, BG328342, BP430683,H51719, AI739661, T96858, BE782390, BU630113, BG025019, BE937466,AA332534, AI343281, AU149094, BP430714, BI087773, AW393255, AU151220,BE076727, AI452809, AW877655, AI003527, AI391554, AW602975, AW877662,AA633915, BF108860, AU149996, T96859, R83277Orc3: BM478060, BU509511, BQ718539, BU153003, AL533919, BQ220405,AU118962, BM550235, BG187255, AU117920, BQ719965, BQ716606, BQ953945,BU166296, AU119182, BI769170, BI819545, CD655888, BX409716, BI520392,BG214275, BI770086, BI091781, BM802602, BG187780, BU170385, BI769508,BX433033, BX507848, AU124361, BX488094, BM785800, BG205874, CD679490,BX490513, CB153029, AW967051, BM826548, AU139285, BG572634, AL533918,CB152486, AU136510, BE536929, BX352629, BQ102522, AW369628, AW449272,BF672680, BE882468, BX343146, BG943457, BF698289, CD702536, BF667912,AU280292, CA842624, BF674961, BF184165, BQ441255, BF794512, CD699734,AL600904, BX462706, BF215571, AL709158, AV753736, AI904063, CB123265,BX486937, BG214276, CD245870, BF059711, AI651375, BQ102253, AL711211,BF964587, AW500090, BM751168, CB963370, BI862225, AA442539, AW887723,AW801684, AV708200, BM152858, H17704, BF683230, BM848524, BQ772810,BQ361466, AL710982, CD242978, H11812, AA305227, AW607564, H94935,BU632641, BX328353, BI255412Cdc6: AL562624, AL521818, BM465884, BQ064897, BX451346, BX349920,BU846236, BQ675107, BI260747, BU633837, BG256606, BM464160, BQ228599,BG252312, BM559225, AL521819, BE907412, BQ070080, BU619893, BF698043,BE741201, AL526150, BG765988, BU173127, BF699051, AA502608, BG165110,AU129648, CA488634, BM450676, BF028885, CB135870, BF185000, BG026757,CA429336, BF977528, BF240966, BG766090, AI478744, BM803439, AA045217,AA813386, BE565947, BF571756, AL710150, BF307679, AA723372, BG721945,AI433558, BF208758, BI559407, BX482661, BF102841, BE779410, BQ441118,N69246, BF310791, CF123750, BI006635, BQ775002, AA907374, BM011340,CA429634, W03300, BM845715, AA113790, BF210909, BM706052, H59204,BI255053, AI424746, H59203, AI052065, BE073887, BE550416, AI341585,BG025851, BF221502, T83032, T90351, BG720011, AI953729, AI699473,BF115521, BF223422, AI808683, BE085836, AI699980, BX355209, BE086769,AW518847, AA099980, BE965778, AA836395, AI766778, BE869748, AI802324,AA584340, BE693538, AI567411Cdt1: BX332414, BQ935210, BU931977, BM811548, BU849056, BQ062875,BQ053758, BU187852, BX406047, AL555432, BQ278148, BX402195, BU845736,AL580756, BQ058496, AL581992, BX332413, AL557066, BU930971, BM016975,BX421258, BU190377, BQ960305, BG824304, AL520240, BM917547, BQ053108,AL582018, BG393757, AL520239, BF791881, BM019024, AL556319, BI092793,BI258203, AL559054, AL527465, AL558613, BU856820, AL524910, AL580393,BX333703, BQ063175, BG259986, BI335580, BG327660, BM556535, BI222927,BG251456, AL520887, BI224536, BI335105, AL524909, BQ053124, BI093258,BG745159, BX405984, AL515463, BE910713, AL518299, BQ054892, BQ053069,BI333817, AL518300, BU189533, BI260243, BF972427, BQ684815, BM695575,BQ652285, BQ649771, BQ647760, BQ647212, BQ645148, BG822442, AL581566,BQ652445, BQ649937, BE544515, AL577935, BX405983, BM927844, BE727635,AL515464, BU176676, BU931060, BU859159, BX366934, BX355022, BM800496,BM463356, BG389325, BU164161, BE388067, BX464574, BE778380, BF237902,BU158281, BM917445, BM809482Geminin: BM550773, AL522354, BI092791, BX375519, BX414734, AL562503,BI861855, BM471496, AL518006, BQ430578, AL580178, BG577005, BG032232,AL525229, AL518005, AL522353, CA417249, AU118695, BQ645204, BG612964,BI855710, BQ718513, BG577324, BI086620, AL558330, BG776051, BG777134,BE893489, BF967933, BG776192, AW996997, BF666338, BQ015308, BQ064691,CD366250, BE910343, BM699599, BG776218, BF029154, BU629613, CB992796,BG825264, BE613337, CB131959, CB049968, BQ772723, BE564333, BI759810,BF666672, BF808421, BF700297, BG337926, BE565866, BG776386, CD367234,BF213350, CB136099, BI830428, BG612435, BE535264, BQ575533, BF667576,BG505022, AA447810, CA442918, BU623074, BF696555, AA393139, CD708137,BF699912, BF967209, BF056288, CA503202, CA312813, BG530534, BF109418,AV756510, BG778341, AA235222, BI093913, BF248391, AI968057, CD686529,BU685799, BG777305, AV734242, AV689368, BF240393, AW006287, AI828103,BF003138, CB049969, BF947954, BF594599, BE048465, BG429246, BG180421,AI803434, AV734302, BE219705Mcm3: BM467763, AL551465, BQ066322, BQ061652, AL559830, BQ059704,BM471050, BU849776, AL545116, BQ063041, BU541430, BU860117, BM542415,AU124791, BU857116, BM453648, BQ056448, BM927480, BQ218351, BQ057647,BQ940737, AU119321, BX462455, BQ898140, CF995699, BI772155, AL549372,BQ214499, BU856617, BM007763, BI223143, BQ652945, BQ649476, BU509755,BQ058522, BQ641758, BQ064200, BG281527, AU133404, BE249947, BU601317,BU154249, BQ927115, BI457651, BX462766, BU558287, BQ051029, BM917594,AU134083, BM561561, CD656673, BQ422727, BQ058080, BM478599, BQ881515,BE795211, BI196606, BG034961, BE892181, BQ649956, BM479437, BG765473,AL527918, BE560376, BI261474, BI599305, BX348989, BE793456, BM461732,BE620320, BE783059, BE799563, BE561200, BQ064568, BE620857, BG681460,BE616575, AU124152, BM832703, BG392301, BG259417, AW083217, BI086286,BQ650935, BI259905, BG686972, CD642696, BI091236, CD655620, BI551396,BE778348, BG773437, BU193733, BE274144, BE891644, AW732422, AU131124,BG742232, BU178300, AU123260Mcm4: BX363316, BM557639, BM479183, BU163628, BX341147, BQ956710,BQ689703, BU855555, BM423607, BQ689028, BQ684773, BU149764, BM917541,BQ877570, BQ962733, BQ213101, BQ679476, BQ931933, BQ670123, BQ680471,BQ878671, BU196152, BQ218770, BQ687458, BQ058022, BU838204, BQ231069,AU124599, BQ060869, AI936566, BQ066067, BQ066435, BU182872, BQ065206,BQ061896, AL710281, AU125558, BM560344, AU124716, AU130095, BX341146,BQ060907, BG683134, AU131502, BM909380, BI259276, BQ676347, BQ054534,BU601939, BQ056963, BQ883247, AU124662, AU134265, BQ681631, BI092911,AU124469, BU151359, AU131979, BU860012, BQ058401, AU126357, BE740475,BG772025, BU154598, BF058934, BQ670493, BI520579, BQ681697, CD643530,CD655257, BQ948077, BE796484, BQ681384, AI738700, BF569146, AU124670,BQ772225, BI117233, AI923706, BX100324, BF059052, BQ652623, AU131348,CF265157, CF594355, BU940867, BG339157, BF116228, BQ682913, BG029854,BG421025, BG248645, BI223223, BE891270, BE741088, AU130533, BG684174,BQ675821, BI830911, AU136189Mcm5: BX446933, BX443180, BU179314, BX465121, BX360307, BQ219621,BQ059059, BX374727, BM560991, BM802651, BU148505, BM478574, BM480184,BX407417, BM558890, BU538182, BX331301, BX465031, BQ893665, BQ671418,BM559170, BX331344, BU156108, BQ645833, BQ069574, BX458285, BQ895922,BQ057750, BQ054136, BQ957762, BX367432, BG767144, BQ065023, BQ055590,BM470663, BU839673, BQ065213, BX368805, BU192073, BQ065931, BQ232104,BM917136, BQ880654, BX346462, BU163845, BQ672003, BQ434878, BQ647973,BG770644, BU557340, BU165017, BG760478, BQ671606, BU541449, BQ670216,BQ649375, BI086963, BQ669996, BI909897, BQ935556, BM043366, BQ642797,BU195081, BQ222354, BU190738, BI869446, BX346537, BQ066237, BG770167,BU557310, BX465120, BX388269, BQ943544, BQ069268, BQ679299, BQ683703,BG576914, BX341163, BM051781, BM719141, BQ440728, BQ431588, BQ643976,BX381461, BU845031, BU839453, BQ213876, BX407118, AU131148, BG685544,BU178502, BQ929382, BU556785, BM457715, BU166135, BM927634, BE735173,BX428497, BX407353, BE253723Mcm6: BM563815, BQ689609, BU178707, BU185218, BM917146, BU180530,BQ691498, BM917702, BQ721374, BQ430793, BQ710328, BQ276415, AU124829,BM551692, BM457121, BQ919455, BQ688139, BQ685964, BQ424418, BM453163,BQ671824, AU143594, BU542273, BU146898, BU178966, BQ072203, BM461535,AU131056, AU133299, AU125636, BU180371, BE383991, BU181929, BQ691761,AU125495, BG686841, CD242701, BG685821, AU117647, BX483567, BM564401,BG390247, AU133321, BI870675, BI084962, BE731324, BI084168, BG532524,AU126102, BM803211, BE734309, BM450955, BG419290, BU146822, BM013848,BG680470, CD643818, BU176030, BM917579, BM045567, BE733405, BG877987,BG538573, AU142944, AU130133, AU124506, BE796828, AU137338, BG253660,BQ879136, BG386500, BM048943, BG914034, AU124893, BX451899, BM012817,BG389994, BG030690, BE731558, BG877979, BE407913, BG878155, BX416717,BE385730, AU128720, BE618973, BE268695, BX118733, BG256582, BG878151,BE513514, BG877982, BG335342, BE281191, BE778969, BM842510, BG878152,BG877994, BU506698, BE280389Mcm7: BX446600, BM916932, AL555833, BM451540, BX342306, BX424231,BQ279230, BM462954, BM468766, BU500250, BM803547, BM557336, BX443366,BQ070647, AL561620, BX355367, BM908241, BQ924446, BQ887320, BM912799,CD108811, BX324854, BQ643995, BM463747, BU183306, BM927622, BQ055649,BQ053452, BX428085, BQ673910, BM921077, BQ887860, BQ883251, BU147232,BQ071179, BQ935246, BQ652903, BQ883056, BQ674104, BQ641811, BQ053620,BM917214, BU162886, BQ052004, BQ891995, BQ878240, BQ953990, BQ063971,BX405959, BQ898941, BQ061151, BQ054401, BQ917453, BM564271, BU189313,BQ058499, BU161199, BM469583, BQ218009, BI522846, BU855416, BQ056795,BQ643247, BQ069037, BQ920442, AU125112, BU194965, BU854868, BU183465,BQ720104, BQ228405, BQ214543, BQ064840, BQ650571, BQ642612, AU125755,BQ956957, BU526752, BE740091, AL561593, BU165222, BE792286, BQ643233,BQ676107, BQ070446, BQ225752, BQ670399, BQ932333, BM810332, CD051232,BU527906, BU942698, BI335520, BQ057726, BM554740, BE799854, AU124962,BM914800, BI825746, BX324853Mcm8: BQ055956, BQ070426, BM454681, BU556999, BM904262, BQ441929,BM559514, BM808018, BM459480, BM808016, BQ070219, BM009484, BQ940417,BU162199, BM558689, BM912457, BQ434761, BI862190, BM810194, CD642958,BG422937, BG338287, BM015340, BI859244, BG762185, BU509003, BG420680,BG023796, BF309111, BM453735, BM466057, CA495297, BF306586, BE513731,BI086506, BM009302, BX504348, BE898012, BG338630, AW955317, BE273079,BG827920, BG396259, BE269095, BM793002, CF137101, BF308208, BM913291,CA425682, AW960988, BX282225, BG168597, BF973469, BE278386, AV645497,AA325061, BE311854, BG339877, BF754616, W94454, CB136734, BI225492,BG434327, CA445505, BG761050, W25728, BM751186, BE842789, BM825974,BU955551, BF127844, BQ007416, CD299273, AI086063, BE928109, BF088599,AA225696, BQ071854, AA226268, AW440309, AA370141, AA193063, BQ334627,AW845751, W94336, AA609373, CA436668, AI609077, BQ320963, AA563920,AI537281, AI200790, BG259140, AI219139, AA192859Cdc7: CA441701, BG170872, BM463748, AL044123, BE789148, AU120443,AU129167, AU116849, AW968900, AW574512, BI462237, AL602215, BM789148,CB959717, AL039323, AA814975, AA936081, BG721963, BU657893, CB216422,AU117631, AA768993, AA131310, BF366907, W76628, N40295, BF982876,AA488999, AW405542, AL044122, BF031756, BQ221549, AA291015, BX419687,BF696442, AA488783, CD523327, BG116756, D20593, CD689440, BG116838,BU568048, CD642993Cdc5: BX350355, BU192616, BQ427813, BQ961587, BI222621, BQ962695,BX331396, CD107746, BQ427606, BE275179, BF982513, BU195085, BX483740,AL558731, BG431157, BM450338, BM925609, BU073210, BG028239, CB306835,AL706102, BG178910, BF025810, BX446071, BU508497, BM718344, BX349125,BQ423785, BM505336, BF977508, BI823054, CD103634, AL135197, AU135978,BF132826, CB160730, AL710914, AI679458, BE617311, BG390164, AW959030,BU933396, BF217466, BG502998, BM894208, AW268817, CF135420, BM146535,BU071659, BE884277, CD101983, BF035463, BG424071, AI143113, AA044750,CF143619, BG722285, BM127700, BG327622, AI122932, BM804765, AV682172,AW954903, AI279537, AL580487, BM894481, BE781164, CK024078, AV762357,BF744457, BM834441, BG121920, BE541230, BF679988, AA811533, AI221677,BG897659, CB052718, AU136923, BU623810, BG497404, CA448370, BF813646,BI048250, BF214089, AU131684, BG540599, BG942273, AA191036, BP429997,AA249176, AL710062, BE140574, AU127833, BG614948, BE140795, AI583919,AI909768, BI918547, BF795413Psf1: BM458856, BU171017, BM450503, BX384069, BQ070512, AW499844,BM151985, AA860312, BF692084, BU430742, AI190765, BQ440331, BM152648,AA725561, AA383128, BM465819, BU659306, BI333600, AA355925, BG910353,BI223929, BF892016, AU099454, AL044646, AI184188, N39921, N39947,AU076561, AL597443Psf2: BU597296, BU184963, BM449472, BM043804, BF683514, BF311745,BE514071, BE513254, BE382866, BQ277667, BQ229290, BG825252, BG772776,BG284180, BG104289, BF795157, AU126087, BF035586, BE796384, BE795838,BE795306, BE561044, BE274253, BE312319, AW249012, CK001498, AL560880,AL560669, BQ233393, BG420251, BE267495, BE258240, CA455226, BE251065,BU957713, BE791539, BE267221, BX415204, BI196248, BG118214, AL529785,BU595469, BF310321, BI257993, BF684568, BE561525, BE251621, BG475509,BG527542, BE793125, BE562088, BG519560, BG475384, BE259285, BU601226,BG339264, BX456910, AV712739, BF312439, AL526847, BU603101, BE260083,BI832397, BU940719, BE255698, BE514978, AA521273, BG469677, BF209856,AL563552, AI828992, AI583174, AL582217, CB112523, BF238335, BG531588,CB129701, BF312015, BE878751, BU625683, AL582077, BE222543, AA262870,AL562756, AW958853, BU506537, BQ361100, AA251319, BM832297, BE296429,AI827298, AL560926, BE907417, BU729618, BE799212, BE268868, BM126492,BE262182, AA053046, AL582250Psf3: BQ231741, BQ948256, BI489800, AL555105, AL524624, BM904357,AL525185, BX406244, AL529159, AL550963, AL524746, BI753591, BI770007,AL561070, BM016893, BG387533, BQ422835, BE782757, BF316873, BF797649,BG765190, BF796771, BM925118, BM722252, BG769825, CF141388, CD676320,BE749159, AL711201, BE297646, AW674872, BQ645203, BM926055, CD693113,BM804294, BG257517, CF552524, BM786881, BF797402, AU142374, BE208552,BF239248, BF310190, BQ890204, BE256868, BG249299, BE907809, W79671,BM754989, BX328153, BU939987, BE281396, CA430225, BQ304813, BQ027991,BM542908, BF769732, BG744402, AA353408, BP430213, BM564422Cdc45: BM550683, BX366266, BX358668, BM478173, BX345270, BX355266,BX366366, BM557094, BM557313, BX358667, BX346442, BX371229, BX352708,BX366365, BX451104, BX352909, BX349664, BX331394, BX328445, BQ069733,BX448615, CA454819, BX448616, BX447114, BX349663, BU184174, BX328421,BQ427880, BX328446, BX451105, BX352910, BX334120, BX409672, BQ214084,BU171037, BQ233704, BX391089, BE747427, BX346464, BX428526, BX422691,BX331393, BX367431, BX367477, BX391088, BX367513, BX325504, BX352709,BX362080, BX367505, BF026159, BE869669, BX325558, BX366268, BE260534,CB124085, BX371230, BG122390, BG387745, BG252967, BG180337, BE897594,BX367410, BM917964, BX346526, BX328422, BQ674776, BM912689, BQ436443,BF965716, BX376594, BG720395, BF125841, BI546622, BQ216400, BU537659,AL711006, CF139190, BU618386, BX367472, BX328725, AI768340, CA454402,AW081615, BX366267, BM751026, BX328423, BE795241, BU618460, BE255146,BG386934, AI369688, BX367409, AW674262, BE903958, AW674908, AA700904,BX389190, CF141215, BE501602Pola p180: BU508486, AL543898, AU121118, CB134498, AU132112, BX327138,BQ883339, CB121808, AW674983, CB149914, CB140712, CB152927, BQ882043,AL570197, BF210579, BE835570, BE818389, AA379019, BE837514, BQ312037,BE837504, AI354751, BM475170, CB122291, AL044294, BE771020, AA355814,BQ351870, AW589637, AA383406, BE717631, R72191, BX117096, AA828105,BF888988, AI261685, BE163167, BE817842, CD000139, CB999470, BF899310,BG926114Pola p70: BU508486, AL543898, AU121118, CB134498, AU132112, BX327138,BQ883339, CB121808, AW674983, CB149914, CB140712, CB152927, BQ882043,AL570197, BF210579, BE835570, BE818389, AA379019, BE837514, BQ312037,BE837504, A354751, BM475170, CB122291, AL044294, BE771020, AA355814,BQ351870, AW589637, AA383406, BE717631, R72191, BX117096, AA828105,BF888988, AI261685, BE163167, BE817842, CD000139, CB999470, BF899310,BG926114Pola Spp1 (Prim2a): AL556161, AL513776, BM546142, AL549894, BX401418,BU187783, BM459297, BU193561, BI523986, BI907286, BG034836, BG215267,CF595567, CA406143, BX280180, BM926617, BF572603, BQ947185, BX404971,BM852865, BM756079, BI547222, BF978626, AL578476, BF747008, BX401417,AL573915, BF745947, AV757142, CF140555, AI557036, T75233, BF745931,BF744295, BM464505, AA465014, T10253, BG183395, BE697488, BG205656,BG209815, BG195945, AA434502, CB113799, BG184433, BF746454, BE766105,AA361880, AA255550, BG191366, BE766167, BE766098, BE766038, BE765690,BE769157, N80963, AI216670, BX114039, AW951150, N80656, BE714429,AA255569, BE843957, R61073, BE714404, BF000349, AA093814, BE538394,T93658, R00642, F12922, BE543709, BF172325, T05292, BQ001605, CA411912,BX455830, BM551302RPA70: BM456944, BQ222582, AU119564, AL576308, AU124434, AU125631,AU122638, BM556841, BQ222302, BM542894, BU177749, BU508590, BM456314,BM466291, BG108961, BX425090, BU633264, BU153418, BU184357, BU517134,BG251944, BG828190, BG764082, BI858388, BG758555, BG035161, BG287240,BF796027, BG036436, BG826869, BX488619, BF971387, BM790584, BG119012,BG765594, BG685852, BE292972, BG761657, BE898956, BE743787, BE897915,BF983057, BF665538, BM792560, BI253949, BG755233, AU125797, BE178302,BI093003, BG120570, BQ218906, BM743518, BM742537, BF698180, BE178464,CD579303, BM848213, BM844440, CF121414, BF344035, BM838207, BE927446,CB121597, BE542431, BF664182, CB115068, BM847443, CD580024, BM848384,BF028723, BM851538, BE927448, BE773962, BE773949, BF751549, BM016568,BQ214159, AA460805, BE764622, AU128580, BG029093, BF082772, BX339968,CB130706, BQ230034, BM711058, CB160550, BE927450, BU501405, BE932015,BE773964, BF699259, BF919259, BE932029, BM541370, BM462468, BE171973,BM845370, BX477548, AL553255RPA34: BX333932, BQ064852, BX442975, BQ069120, BQ943330, BQ063763,BQ674220, AU118399, BQ059648, AU143441, BQ673815, BQ439053, BQ278675,BU856528, BE741729, BQ066157, BG825398, BQ668543, BI600038, BQ641985,BI757393, BG333934, BQ054635, BQ070050, BQ066715, BI518754, AI419040,BE271646, BQ063545, BQ058415, AI890508, BM543895, BG336979, BE898769,BI818496, BG421195, BE901546, BG469742, BE887147, AU134052, BI756891,BF308713, BM312218, BE902956, BG433978, BG334708, AI744901, BG254134,BU943380, AI929664, CA488595, BE298500, BG826547, BE394497, BI599375,CK002534, BG779099, BE313107, BE298150, BG501316, BG826213, AU129936,BQ059808, AU126353, BI113916, BE297131, BG424340, BQ642824, BI193274,BI546749, BG716673, BQ055902, BG777777, BG428439, BE019650, AV762431,CD687322, CA842220, BE898527, BE294795, BG616118, BG615827, BF791819,BE568731, BQ059622, BQ054654, BG436837, BF686542, CD702944, CD710078,BG479643, BE898609, BM698831, BQ924421, BE394931, AI961707, BX283385,AA641800, CB145745, BF692608PCNA: BM464765, AL547405, BU162573, BQ233597, BM923901, BM475636,BM809424, BU506972, AL549034, AL549068, BI254350, BU187589, BG686220,BQ716438, BM477662, BM542830, AL572455, BM474328, BQ231284, BU195180,BU161781, BG774625, BQ681114, BQ649204, BU634227, BM474327, BI765443,BM979950, CD519986, BU626265, BQ682146, BI767353, BG707111, BQ679867,BG755768, CD367344, BI254540, BG166783, BQ014636, BI598197, BM977646,BU624262, BG686801, BM016212, CA442951, CA443088, BM976306, BU628431,BQ009665, BE889822, BE738456, AA910951, BQ218579, BI829094, BI226337,BQ050978, CD238945, BG503955, BE888544, CD367010, BQ429019, BM466077,AI348072, AA843679, BE739511, BG540339, BF685141, BM842748, BM829821,AI25272, BU656120, AV717345, BU154500, BI831672, BG533644, BM850147,BQ016237, CB529827, CA446890, BQ447329, BM781704, BG503385, CD364739,BG614065, BG290688, BQ681737, BE887284, BE883191, BG613869, BE746433,CB529409, BQ016228, BQ003193, BG502601, BX473856, CA443057, AV649575,BM995025, BU154811, BG532459Elg1: BX435523, CD643489, AW976468, CB161634, CB051111, BE551573,AW514252, BE042824, AI621250, AI623298, BF669931, AA651909, AW450012,BG389184, CD644045, AA972691, AU149697, BU509262, AA724028, AU126948,AI656767, BI094506, BU428574, BF243394, BE886708, BX102408, AA744478,W87913, BF212165, BE834403, AA857981, AA136031, AA703271, D29036,BE152409, BX109066, H56423, H68973, BE005696, BX461696, AA610813,BE834436, BM312382, AI078312, BG207827, N90506, BG197401, BM724358,CB051112, BE148289, AA806690, AW978010, BX108248, BX104136, AL712199,BG619264, BE163388, AA976805, AA707097, AA705010, AA702235, AA436301,AA436174, H59615, BU682299, BF993160FEN1: BU538692, BX397634, BQ058498, BX424210, BX333531, BU170538,BX443166, BU860300, BQ888965, BQ880548, CA489528, BX433300, BM015629,BX448621, AL560007, AL531350, BX331605, BU535646, BQ957039, BU931957,BX445725, BG828048, BU167885, BM561765, BM560757, BG575417, BU931950,AL519300, BQ918754, BM546237, BX425258, BQ642352, BU538026, BQ641309,BU178840, BU553925, BQ690414, BU539094, BG574950, BX448789, BG337603,BM552061, BQ053379, BQ424018, BF686180, BU176039, BG676364, BQ064038,BU170972, CA454699, BU859837, BI3116779, AL560377, AL560395, BE793493,BG576479, BE792164, BG756459, BG773958, BE311755, BE796307, BG474425,BI767742, BX394237, BM542385, BE795541, BI827898, BU931956, BE799080,BE397382, BQ050062, BE796569, BI3117469, BQ227585, BI3115669, BE780262,BE274648, BQ946363, CF131987, BM917670, BG825257, BU189559, BM803891,BU856251, BU165752, BE794075, BE793759, BQ777102, BI256835, BU541327,BU152651, BE799325, CD243456, BQ278519, BQ219034, BG287218, BI334366,BI116455, BE798996, BG472198DNA2: BX390869, BX329314, AL527195, BG289876, BI869219, BX384719,CD644575, BG106738, BE866952, BG117032, BG036343, BG177711, BE748018,AW134972, AA284382, BG501340, BX384718, AW369063, AW369067, BU658975,BF213278, AW367239, AA282895, BE085640, BQ435874, C20980, BG944343,CD514528, AA974495, AA830575, AA767191, AA748680, AA282803, AW977920,AA732685, AW367310, BF089037, AI186294, AW361984, AI940759, AI940744,AA812151, BF357542, AI248069, W86421, AW378978, BG961093, R05855,BI091087, BI091081, BG505976Ligase1: BI916625, BM555654, BX325045, BU154275, BM044202, BM548700,AL530699, BG743952, BG678604, BU168385, BG774713, BM015149, BG825382,BG681554, BI755126, BU152699, BU543078, BG744633, AU120968, BX329161,BE747873, BM472230, BG257399, BI856896, AU143382, BE794374, BE744087,BM763360, AU120985, BG327553, BE512655, BE873444, AL570759, AL042689,BG747144, BX325044, BE047619, BQ072622, BF038182, BU159409, BM013639,CD579385, BM794429, BF529953, BQ923144, BE297514, BE263744, BQ648677,BG257587, BX362172, BG251839, BF205184, AL558263, BI765243, BM917641,BE512703, AU128254, BE294485, BQ644838, BG024771, CB109099, BQ654248,AA306774, BM711430, BG177788, BX370252, BM846234, CB270211, BI025622,BE257136, BM975458, BM819487, BE266691, BX475022, AL530700, BQ231386,BQ071175, AL710126, AL705975, AL705915, AL697933, BM749091, BI463850,AL602262, BG333926, BG116773, BE294757, BX362173, BM456382, BF797607,CB121210, BM458469, BX503423, BG685986, BM836632, BG469591, BE907368,BX475021, BM793401, BI829665Pold p125: AL578715, BX382861, BX366475, AL560083, AL556466, BX366474,BX402885, AL525375, AL514720, BX350425, BM479873, BM905305, BQ920464,BU855691, BQ054258, BU527550, CA455120, BM008549, BQ070749, AL559084,BQ688129, BQ068026, BQ955289, BU542295, BM048573, BE311672, BQ958499,BM044191, BU859048, BI859768, BG744446, BQ936440, BU173332, BU931011,BG826841, AL580780, BU527075, BQ918345, BE737103, BM008621, CF125207,BG745091, BG472420, CA455000, BQ890540, BU154168, BI334420, BF205093,BG340726, BE798460, BI118205, BG683283, BE547846, BU185961, AL514719,BQ071299, BG258722, BE796517, CD101690, BE274988, BU844535, BE298157,BQ953920, BG390567, BE731346, CD615429, BG029434, BF346914, BG911621,BG281172, BU944555, BF206631, BE513504, BE391305, BG832159, CB321982,BG749237, BG285702, BQ343533, BF312202, BE901507, BU553455, BF304095,BU501677, CD615427, BI227211, BG120642, BE255898, BU161275, BM012106,BF529600, BQ232039, BU539859, BG115290, BM471494, BM742222, BF203965,BQ071659, BE514532, BF689201Pole Pol2: BM799918, BX368245, CA489133, BX452508, AU124277, BX448917,BI524150, BX432893, BE613576, BX400486, BX448918, AI341337, AW629043,BX444300, BU617156, BF029073, BX280062, AW974329, AA448761, BE966475,AA709119, BX400487, BQ318645, BM784534, AI039222, BQ946037, BM454666,AW439589, BE782680, AA282380, BU742406, BM193890, AA448664, BM665497,BF738758, BF766844, BG473220, BM751106, AA333178, AW139478, AI636255,AW242762, BF766936, CA941526, CA941235, BM509588, BF766967, BF766969,BX384417, BM505194, AA812343, N53947, BF766934, BM751342, BG195865,BG185004, BX414432, BG219868, BG210945, BG207825, BG197398, BG193392,BG188146, BG195403, BG190325, BG207824, BG216817, BG216115, BG212524,BG209425, BG196918, BG190326, BG189205, BG188145, BG218864, BG203607,BG182912, BG215183, BG220834, BG216818, BG203604, BG195863, BG194393,BG194392, BG181396, BG212527, BG197926, BG195866, BG189206, M62099,BG214127, BG193903, BG192860, BG212525, BG207823, BG207822, BG204615,BG202577, BG202576, BG202042Pole Dpb3: BX471071, AL544919, AL531155, CD171731, BX422049, BX403356,CB159628, CB152302, CA487866, BU956441, BU931411, BU844651, BU844620,BU193214, BU181445, BU178251, BU160282, BU153515, BQ956965, BQ932794,BQ896428, BQ883962, BQ691435, BQ688656, BQ643218, BQ437007, BQ425615,BQ421168, BQ227667, BQ224773, BQ220928, BQ057666, AL713425, AL711259,BM853361, BM830557, BM818099, BM811580, BM552527, BM478816, BM474837,BM465332, BM463844, BM451660, BI463584, BI224290, BI091613, BG3181075,BG111071, BF978613, BE895839, BE883232, BE872164, AW246427, BE910559,BU161793, CD300569, AL542290, CB132298, BQ924495, BM920044, BM847093,BM451747, BM013495, BI668995, BG700033, BE781043, AL598822, AL550727,BM749328, BM477218, BI755256, BG720455, BG505578, BG387715, BF983616,BF978547, AL541402, BM557688, BM558417, BM193306, BG024009, BE543436,BI333822, BQ059205, BI561738, AU280159, AA524279, BM753932, BM014466,BE880199, BE242720, CD710143, BU959989, AW136187, W03622, BU571151,AI634435, AI991485, BI334810Pole Dpb4: CD674888, BU597500, BU595433, BU594966, BM924454, BM555016,BM5511010, BM009306, BG491874, AW081785, BE910607, CD107195, CA307504,BU520765, BQ233876, BM912894, BM809080, BM727074, BF237493, AI554783,AI436367, AI886832, BU597812, CA454961, BM929605, AI432454, BI667558,F26406, BQ954219, AI815728, BF025828, BE276764, BF764960, BM725423,BF107426, BG760830, BU740914, BG740141, BU077279, BU963250, BF237693,BG683544, AW970445, AA927473, AI142293, BM714678, BU739305, BM984649,BI599890, BU076938, AI797479, R07547, AI188727, BG682813, AI815926,AA513753, BP431280, BU537093, H27059, BP429067, BM677848, AA811357,BQ219306, AI833007, AI090223, AW368694, AI148002, AA676886, AA353038,AA740345, BQ640428, AI191303, BQ013037, BX112032, BU953216, AW955899,AA400317, BQ011449, BM688755, BM687694, AA368986, CD693537, AA400632,BF944449, BF378717, BE615920, BE408046, BF978324, BM723287, BM682293,AA536076, AI336523, AF202331, BG461940, AA639692, AI970899, CF529348,AI017725, AA468753, AA978356Topoisomerase I: BU175449, BG574241, BQ918804, BQ720771, CA488073,BG506927, BM788013, BG546269, BM722996, CF137671, BX403047, BX391491,AI878932, BG493034, BU934394, BF977810, BE748187, BQ230349, BE733657,AW025108, CA487823, BE070282, BG401860, CB242988, BF912374, BG433599,BI561949, BG252538, BX406161, BI092973, CB959389, BF887734, CF145440,BG529331, BF594476, BF726053, BI087263, BG540279, BF002422, BF214159,AA765988, AA594329, R60159, AW368554, BF768633, BF923424, BF573926,D55538, BF105824, BF741104, AW854287, BF095014, AV708869, AI493041,CF127017, CD523275, AL559809, BU625720, BU195531, BQ950231, BQ718893,BG778556, F07589, BI834633, BU940860, BG169393, BE818064, BQ438538,BU429936, AI271876, BX389156, D54890, BX391490, BM541278, AA887955,BM699908, BF216295, AW368250, AW368275, AI479910, BG532987, BM720782,BF342838, BQ379859, AW003919, AA639463, BX403046, BE172121, BF924434,BG942263, BG611737, AI337284, AA987503, BF030802, BP430593, AI637947,BM985011, BF694314, BG569753, BF887735TDP1: BM545366, BX357935, BX352942, BX368062, BU185781, BX336700,BQ214685, BQ233509, BX367994, BX368251, BQ689475, BQ049211, BU163540,CD654830, BI253420, BX388603, BX357934, BG291484, BQ277263, CD642861,BX336701, BU174608, CK000808, BE894450, BE613472, BI490906, BX352941,BM150331, BQ223905, BX472747, BX363837, AU136908, BX407827, BX461234,AW968944, BX368068, BX474790, BE387073, BE614223, BE786331, BI222338,BU429540, BG772310, AU135919, AW249271, BX367862, BU849335, BI822990,AL602103, BX477415, AL598723, BI861569, BI489958, AW962673, AL705760,BE909004, BE747879, BM712636, CA425849, BX401951, BE312937, AW849814,AW849937, BG475315, CD641965, AW007897, H49893, BQ010512, BE246145,W76100, AA332235, BF196744, BM462605, AW410205, AI480141, CD703683,BE247287, AA609339, BX475227, AA477148, AI209111, AW961554, AA330280,W72865, BU738356, AA336839, AA514317, AA620407, AW000979, AA504522,H49894, AU156926, AW129282, AA628378, AW589860, AI636696, AI989590,AA716609, AA489121, BF896143Ctf18: BQ231004, BX447012, BM806765, AL562324, BX371387, BU553656,BE795677, AL516520, BQ962210, BQ650965, BE797877, BQ645686, BU845747,BQ646300, BE898071, BE901267, BU509771, BQ650789, BI457170, BI196074,BG481033, BI823171, BU625412, BU633872, BE888887, BI766695, AL516519,BE902046, BG168881, AL524240, BM674122, BQ648925, BQ651106, BQ646373,BM715777, BM127632, BF308850, AW973666, BG761379, BU845239, BM832965,BU616468, BF306837, BQ645867, BQ773121, BM127327, CA421425, BE262702,BU159091, BM703773, BM793661, BF513105, BM005987, BE314193, BF347314,BM906352, BE313266, BQ644028, BQ650233, BQ647544, AI831961, D61532,BG480239, BX117345, BE780529, AI650845, BE300859, AW196692, BM150350,BF093805, BQ367862, BQ073027, AA478378, AI824849, BE279389, AI620989,BU540454, AW236312, BG825945, AI918000, BE242499, AW662226, AA352175,AI355547, AI916173, BM701214, BF182688, AA610722, AI276362, BQ651661,AW149595, AW631061, BF434726, AW467884, BF091910, BG488804, AI401116,AW904596, AI689357, AI382635Scc1: BM466374, BU146139, BU164770, BQ434145, BU190012, BQ222984,CF552154, BM474979, BM477931, AU130565, BU156118, BX390631, CD657694,CD654104, AU123599, AU131556, AU123557, BM927599, BI089741, AU124372,BI869474, BU625171, AU137268, BM920285, BM479826, AU138617, BM803806,BG779064, BG503734, AI905425, AU134242, AU130905, BU175990, BG289967,CB959416, BQ437529, BE870127, BU940947, AU135199, BQ229673, BF797759,BG254176, BU178041, CF619358, BM452576, BI093343, BE560508, AI627668,BG390625, BQ638398, BQ230670, BU509303, CD358988, BQ574279, BE867847,AW028126, BQ230181, BU181371, Z78332, BQ218011, BU598145, BF103682,CB143108, AL705581, CD557703, AU134649, AU125960, BM833822, BM749176,BM452530, BG505923, BU431249, Z78334, AI739002, BF540787, AU128854,BE895809, AL540173, AU133303, CF135927, BM907180, AU129400, BF091717,AA129353, AL558080, AL046011, BM833939, BM467920, AU135442, BF794442,AW500227, BM461566, BF590668, BE748270, AI017447, BI093513, AI367597,CB143109, AA699622, BM478563Scc3: BQ946254, BQ224497, BG678247, AU131359, AU141951, BX643586,CA488740, AW993480, BG284625, BM468092, AL042846, BX506229, BI223205,AL582073, BQ229101, BF796496, BU431562, BG682345, BQ718426, BG114650,BE871224, AU132652, BE929374, BM799307, AL701691, AW499961, BX505839,BQ422046, BF085120, AW966123, BF085121, AW993214, AA311870, BE817052,BU430955, N25477, AW937839, AW501973, BX503773, CB988449, AL701626,BE541958, AL710194, BE817053, BF085130, BX505398, AW892743, CB963696,AL582049, BX643342, BX643410, AA179766, BI088302, CA454732, AL708036,AW999070, AW966631, BF367226, BE540683, AI064692, AL692142, AA334313,BG698257, BX473973, CD238866, BQ371413, BF330420, BE832149, BU431563,BE832148, AA385639, D78828, BE844082, AL710458, BE844095, AW993032,BX473971, BM751899, BG951313, AA249600, BG187519, AL603592, AU117247,BX102249, BX646186, BM718664, BX474425, BI771735, BM833941, AL600231,BE708022, BG536049, AI351861, BG899130, BF830533, BX437683, BG167708,H75808, BM561699, BG256800ATR: BM452469, BU146099, BU193242, CD359676, AU133155, BQ226453,BU616550, AU138930, BU521017, CA771525, BG679313, BI259481, BE894977,BX476619, CB134903, CA771217, BU620031, BG770191, AI685264, BE221326,BU676069, BQ432546, BM855140, BX646290, BX476608, BM141700, BX476618,BM129429, BM129718, BM141963, AW769028, AA453176, AL707012,BG768017AW029178, BG960271, BE646363, AU154536, AA746485, BQ025557,AW769551, BG960875, BE091396, BG026395, AU157822, BM796532, AI584172,BU431210, AI288527, BM459025, AA731840, BE859077, AL039634, AW976047,BF222914, AA825525, AV751232, AW390089, AW152454, BG392173, BG223235,AA551327, CD644383, BF094478, BF930497, AI962936, AI871554, AI279279,AA837410, AW978820, AI394218, AI285634, AI280393, AI127664, AI078770,AI027417, BU076885, BX497770, BG192205, BG189101, BG184889, AI088580,BU077225, AA215661, BG208865, BF110182, AW237573, BG210328, BG221227,BG201951, BG191706, BG182288, AI902747, BG194892, BG204028, BG208221,AA747410, AW390065, CD642306, AI689705Chk1: BX384024, BX425856, BX352948, BM458297, BM803862, BX363020,BX384025, BX383978, BX363830, BX443777, BQ071454, AL515222, BQ919396,BX346314, BX363829, BX414303, BQ424951, BX345096, BM048703, BM478961,BU620586, BX386787, BX386786, BU181250, BG717056, BX440542, BG687019,AL523644, BG258170, CA441277, AI924526, BI088504, BF795495, AL708308,BG612596, BQ226720, BM968823, BG339614, BG944287, BE299090, BI521358,BX351192, BG828404, BQ223060, BF310022, BQ641604, BM558032, BG218896,BF242017, BF001625, BG470645, CB127107, BG192348, CB124258, BF204894,CB124369, BE464453, CB125201, BG256454, AL559804, BE298964, BX383977,CB124285, BG194802, CB998143, BE904400, AL559805, BG216281, BG191840,BX425855, BG215785, N99369, BG470702, BM193374, BX363019, BI197298,BE882051, BE297644, N53057, BE548526, BI824209, BG211479, BX346313,CB142976, AA224307, CD694666, BQ322635, AA962684, AI536947, BM455102,R86187, H67490, BF973418, AL515221, BF946916, N71469, AI750793,AL523643, H59530, BU927896NBS1: BM542698, BX405940, BG182890, BU166634, BM461758, BG214621,BG388866, BG284646, CF593314, CB1123692, BU517247, BU661996, BM014420,AW976050, AI796269, BG483074, AU118357, BG109073, AW978306, BG392111,CF994271, CB250418, AW183153, BF027776, BU620472, AI888159, BE694454,CB989468, BF511289, AL713597, BG292394, BI962748, BG202556, AW363125,BF028917, A767797, BE142989, AV715636, BU686090, BE695861, AU144944,BF219376, AI478631, BF208284, AW237021, BF217323, AA535147, AL041061,AA741007, AA577530, N22869, BE694368, AA713939, BF222791, BE892618,BQ354782, BE566896, H98655, AW391193, BE694374, AW340253, BG197194,BF062731, BM835126, AI890179, AA807181, AW025671, AW593423, BE089552,BG194661, BG194211, BG187424, AA463450, BG184671, BG209170, BM833754,BE694416, BG198067, BG214402, AI478521, AA835830, BU172525, BQ380443,N36514, AI858133, AA907134, BF096050, N51586, BG196671, BU429506,BE694353, AI952672, AI377839, CF137847, BE142840, CB135538, R48068,AA535711, AW207441, BF219034Hus1: BX510134, CD520767, AL554895, CD104810, BU933524, BU932644,BU600981, BU171912, BF185772, BG386353, BG286955, BE874516, BM462752,BU932377, BE568470, BE892098, BM546627, BF510091, BE543378, BG330719,BU193379, BG703514, BM906889, BE644764, BF796878, BM822628, BE566605,AA902233, AA280710, CA418558, BQ646867, CD678464, AI675254, AI968159,BG028551, BU680921, AI968626, AI750426, BU784416, BE891273, BF056974,AW518029, R29753, AW270395, CD357688, AI656993, BX115181, AI149713,AI538328, CB992757, AW965692, BU588470, BF222727, AI654498, AA693873,AA353895, AA828114, AA773515, AA897773, AA652723, BX500811, BF998283,AI968739, AI656972, BX370241, AW467865, BF211281, BU928301Rad1: BX439078, BX362814, BM915064, BU508168, AU142492, BG325636,BG254417, BE739684, BU942019, CB997499, BG528822, BF305274, BU192569,BE379759, BX472344, BM453151, BG502187, CD365064, BI821006, AW779759,CD370483, BU567700, BE542464, BQ277303, BF381656, BF103945, AL697883,BG687436, BU431185, AI732815, AI870850, BE866735, AA486301, BM875591,BX362071, BU623683, CB126098, AW237104, BG687442, BF084168, BE565545,AI052547, AI628587, BX105685, CA432131, AW373219, CB306975, BU682777,BM875344, AW473643, AW473637, BM790461, BG777245, AW104439, AW001011,BF667027, BQ214495, BG108349, BQ433094, CD513520, AI685362, AI075030,CD672667, AA968417, AA029300, BF807890, BM264116, BG036263, BE843169,AW779236, AW819703, BX373966, CB218092, AA768474, BF242093, AW579006,AA464502, AA227739, BE930194, BF375637, BE549430, AA913007, BI862650,A871190, BX369672, BX369671, AA228124, BF748944, AU157657, AU123712,BE928477, BX435862, BM015762, BE645342, BE379989, AI885817, AI734194,BG612563, AV711443, AA464501Topoisomerase IIIb: BX424738, BX425419, BQ651682, BX403442, CD107506,BX446190, CA453925, BX346535, BX418504, BG763535, BM549973, BX403443,BX388297, BQ649447, BQ279059, BX353337, BX428354, BM562369, BX389266,BF690073, BM922961, BQ884077, BI254650, BG751464, BF348239, BG767127,BX375918, BI199856, BQ425197, BX333417, BQ939934, BG748897, BX418503,AI361851, BQ183439, BX428593, BM719837, BF689997, BG827105, BG180123,BX431485, BM020904, BX431486, BG251915, BF568363, BX430251, BM982636,BM542563, BX353336, BF839819, BX389265, BX456723, AW082912, BU739692,BM668058, AA581879, BM929485, BX375917, BF569140, BF840332, AW081400,BQ072301, BI908256, BI458744, BQ437166, BE262344, BX471363, BX333418,AI653725, BG281858, AI432376, BG910301, AI271458, BQ018902, AW594115,AW580188, BF683888, BQ052367, CB958279, BU733478, BU956077, CD656636,AA576862, BX425418, BQ925386, BG519835, BQ267454, AA789096, AI884361,BQ270362, BU194029, AI654571, AV724647, AI797309, BX088825, AI252649,AI368666, H30621, BG281907, BF971171Rad6A: CF242862, AL556664, AL554264, AL551212, AL545489, AL527666,CD245975, BX420488, BU177002, BU176550, BU158774, BU155911, BQ938012,BM904536, BM671147, BM547988, BM460782, AL601372, BG477320, AL557798,AL552044, BU594647, BQ773667, BM127839, AU137774, BE873022, AI126625,BM128123, AL547443, AL547028, BG771586, AL561578, BU596427, BE893452,BG037200, BM128046, BM127780, AU135850, AI912983, AW051875, AA917931,BM172179, BE280929, BE504240, BF308088, AI367248, BM888354, BE465165,BI914734, BI562526, BG426078, BG399765, AU128974, BI117499, BG709332,BI223209, AU123986, AU126997, AL540766, AL545955, BQ717080, BX377743,BE276997, BG429673, AI830472, CB118540, CA398015, AU280101, AI367259,BE001808, CD673205, AU128994, BQ050943, BE934281, AA314005, BG249012,AW206875, AI984287, BM752230, AI097110, BF030505, CD299140, CB962261,BE867709, AW205767, AI371888, AI087376, BG680605, AW139418, BP431686,BM741851, BE002061, BF694346, AA442497, T80555, CB127128, W77761,AA340148, R92832, AA808831Rad18: AL515920, AL525404, AL562493, AL515921, BM479176, AL519429,BX327634, BU633444, AU130305, AU124369, AL519379, BQ878144, BX118224,BI260485, BX644573, CD657250, BQ002046, BG403172, BG187245, BG687139,BQ438688, AL519378, AL602096, BG501779, AV689196, BG434615, BX506345,BG528199, BU928268, BM827802, CB146761, BM783792, AU152279, AI674134,BM987526, BF062100, BE538599, AW024863, AW188470, AW852547, AI140776,BM739442, AI140772, AA625471, AA628928, BM820601, AA953817, AV689200,AL600012, AV689197, BI463280, AV689198, AI075759, BQ012909, BU588168,BE245247, AI826396, AW274711, R59255, AA311754, AI266146, BE715962,AI051483, BI060361, R59197, AI292169, BQ305165, H79432, CD643305,AA972797, AW607437, CD709390, BF241055, R42938, H79318, R17601,AI262720, BQ013328, AL044563, AA494524, AI536060, R18043, R13366,AW804426, AW607158, AW969432, AA745596, R40881, BI060362, AW804432,BG992485Ubc13: CF130960, CD710574, CD692115, CD673025, AL543503, CD245362,CB988950, AU280192, CB215753, CB161684, CB161357, CB159339, CB158868,CB147566, BX110715, CA453274, CA310015, BU959707, BU942968, BU941352,BU935537, BU787955, BU509214, BU195662, BU177345, BQ670955, BQ651546,BQ438057, BQ434581, BQ431772, BQ278353, BQ233659, BQ233603, BQ233591,BQ220048, BQ212232, BQ071631, BQ053849, BQ053589, BM927363, BM920802,BM916123, BM810273, BM805692, BM548060, BM480191, BM456876, BM449746,BI830293, BI829065, BI822117, BI753449, BI603033, BI193140, BG759142,BG758336, BG720632, BG716212, BG701931, BG613290, BG548397, BG531270,BG503962, BG432626, BF974186, AU125145, AU119879, AV758049, BE747116,BE266994, BE314665, BE207615, AW246428, CD685196, CD558824, CB987518,BU940976, BU177313, BG715594, BG715088, BG714343, BG701027, BG615565,BG504869, AW950789, AW250538, BG716471, BE262841, AW673494, CD686244,CB957396, BI197667, BE313357, CD701933, AL583561, CD385216, BU596184,BX415171, BX400510, AL534723FBH1: BQ668450, CD518455, BM475590, BQ073711, BX350417, BM556786,BX385835, BU171774, BM462614, CA976039, AL555827, BU527061, BG682347,BQ932104, BU184837, BM811347, CD513292, BM051895, CF552522, BF792094,BQ953076, BM469767, BU856754, BU507355, AL580250, BQ672631, BM541777,BG575794, BG396523, BM019265, BI462395, AL578234, BQ710339, BQ889679,BQ706264, CF125476, CA439526, BU535409, BM916651, BM014236, BF683805,BG104902, BG385761, BX369664, BG483429, BQ722509, BQ894882, BI3028476,BG422497, CD722593, BE253172, BX483033, AL558429, BE513039, BG117837,AW369165, BQ437287, BG479586, BG830784, BQ129343, BM686491, BX117109,BE541008, BE736140, CA393619, BG109986, BG913455, BM717015, BX356080,BG323256, BF684368, AA045149, BE730963, BG682808, AL044721, BF349688,AW964614, BQ951138, BQ325260, BI006637, BM707421, CB113655, BQ898291,BE378693, AW963805, AA430290, CB159047, CB161429, CA438158, BX364723,BM707309, BM699811, AA428015, AW963835, BQ129349, CA395354, CB321675,BM798328, AI459539, CA945183Mad2: BX092337, BU509241, BQ959603, BQ440642, BQ428342, BM472395,BM472304, BI766194, BU508933, BG532327, BQ425846, BG702724, BU509710,BG614828, BG505458, BX443383, BU963707, BX325759, BU198649, BM016150,BG503527, BX404037, BG679723, BG533781, BX449727, BX346251, BE270292,BU177716, BU662541, BG530972, BG531198, BG116166, BG527529, BE886793,BF305710, BU928412, CA489378, BG503886, BE311763, BX401098, BG496604,AW950858, BE778450, BE270518, BG613007, BF034523, BG504712, BG249673,AW411207, BG501915, BF219704, BE890707, BU598703, CA489522, BG504001,BF130567, BE296423, BF030667, BG531869, BF694258, BE295856, BQ277112,BE895923, BF666701, BI560148, BE543883, AW674988, CD700655, BU659357,BG169697, BE960883, BM458351, BG615578, BM837848, BG506388, BE870543,CA488467, AA490658, BF184132, BE567312, AV715949, BF667164, BG284883,BF666681, BM450737, BU661109, CB137773, CB137684, CB134844, BF698236,BU158230, BF701297, BF240809, BF185562, BF696854, BG613188, BE738000,BG290170, BF696888, BE270517XPC: AL537156, BQ898206, BM556322, BQ918948, BU506961, AU125870,AU130697, BE260062, BQ892451, BG751164, CD643621, CK000090, BE730655,BF981364, BG748625, BG341433, BF972749, BG338028, BU602325, BF317427,BF306190, BI255928, BE278952, BM461420, BE733920, BE252615, BG752811,BI670281, AU120699, AU130155, BE254313, BX474915, BM729318, BG340238,BF314903, AU280283, BG489139, AA287404, BG337505, AU127391, CF995178,BG335426, BQ308142, BG259049, BQ649424, BQ307301, BX505750, BE260137,BF683997, CF140093, BF685974, BX470382, BE257840, AI123414, AU150414,BQ477814, CF141168, BM827376, AA657557, BE221715, BG571695, BX475123,BF090364, BF207269, AL709045, BM856351, BX497971, BM708556, AW504862,BM833387, BG178613, BG620310, BX283619, BG749233, CB269927, CB267080,BM700758, BX486869, AU143301, BX493543, BM852149, AU128095, AA190694,BE702371, BE074001, BX644722, BG396899, CD250721, AL710884, BE262208,AW903238, BF827957, BE766460, AV736879, BG116273, AA329947, BX476805,BM454293, AV734541, BM908255Rad23A: BX386817, BM555668, AL556689, BM923938, BX462941, BQ226301,BX383110, AL518853, BM563676, BX346368, BX443456, BX458814, AL560403,BM800629, BM546406, BM450093, BX400223, BM905361, BQ067487, AL538737,BX448989, BX376642, BU182138, BU501586, BM460305, BM455101, BM553961,BX405327, BX439481, BQ959921, BG397266, BQ231221, AL528006, BQ649073,BU161613, CD516432, CD300604, BU161547, AL527519, BU170588, BQ922155,BM922503, BM806531, BM811343, BQ878719, BX416323, BQ431936, BX424587,BQ231191, BM553778, BU166711, BQ068184, BI3115640, BX336880, BE743148,BU902824, BM543659, CD517476, BX440266, BM928421, BE793785, BM805413,BU663811, BM767302, BI771571, BU543634, BI488410, BI831370, BG828123,BM702259, BG826357, BQ671655, AU120562, BI756007, AL554211, BG575011,CD359531, BI092253, BQ923295, BG765676, CF146536, BE254829, BM764880,BI768800, BE254847, BU186368, AL548714, CD580418, BQ430734, AL708410,BU598395, BU160883, BI770418, BI458425, CB243750, CD300680, BE296271,BU624195, BE792673, BF982409Rad23B: AL542437, BU508207, CF242874, BI761813, BX385070, BX344701,BX397027, BX406219, AL544467, AL540969, BG617563, AU135170, BM785167,BG681545, CD516576, BM846302, BI094479, AL532383, AL554483, AL549735,BI524081, BI086980, AW747914, BM843069, BF949974, AL570682, BE166667,BI460482, BF696085, BG290212, BE018477, BE566434, AW117407, AW631016,AW629978, BM919879, AL516156, BE218017, AA460535, AA305019, BM845855,AW610521, BQ927520, BF082151, AL571657, AW080867, AI221288, BF239610,BF116028, BE218477, BG035090, BF238781, AW610520, BE926034, BG718045,CB995306, BM705797, AL569427, BX411441, BI868477, BF817189, AA316654,AW821231, BF433951, BF906503, AW578686, BF762435, AA932185, BG958581,CA406436, BX387819, AW389400, CA389879, BU959168, AI261824, AW991331,AW663949, BF374886, AW770657, AI703064, AW510997, BE925443, BE220051,BE674734, AW389398, CF553075, BF088680, BF762431, BF692478, BF111238,BX422214, AA359699, BF994844, BE613559, BQ929186, BE817876, BM480327,BF755677, BF795639, BX500679CSA: BI918304, BM833676, BG611935, BM017684, BG722970, BG612963,BI458951, AI950957, BI601669, BU603353, BU533681, AA454500, AW954940,BI828404, BE540951, BF244952, BE567160, AL691658, CB160846, BG387575,AW388466, AW388282, BF206366, AW301277, BI850241, BF665074, CD687697,AW409745, W19086, BF790869, BM147057, BF665145, BG616128, BE568475,BQ218876, BX116922, AA129369, CD109410, BG032140, BG037177, BF588485,BF000147, CF552572, AA159858, BI561029, BM835908, BQ645232, AW418819,BF247700, AU100233CSB: CD653749, AL039860, BG723092, BX644251, BX474980, BF508753,AL702189, BM759548, AL039851, BF094116, BG121679, BI020594, AU185158,BE763975, AV725351, AI418429, AU185476, CA502920, BX485503, BE841244,AA305555, BQ015647, BG259982XPG: BX370344, AL537284, BQ215712, BM461711, BX383623, BI518401,AL537285, BG754702, CD243930, BU680238, BG574639, BQ002437, BI836225,CA503022, AW044617, BQ045373, BU608348, BG282989, CF619292, BQ014611,BI091747, BU624982, AA843311, CA418268, BM793974, AW772514, BU617777,BQ186957, BG400427, BU732780, BU608324, BQ221301, BM504121, BG391687,BG286779, BI711387, A680931, BQ775943, AI417946, CA424453, BM875436,M797308, BE170510, AW317068, BF576042, BE552270, BM507072, BM750705,BM675983, AI885477, CB142083, AI623400, BQ614576, BM830049, BM875687,BE350942, AW854025, CD579376, BM677102, BF360483, BU740308, BQ002355,BM504348, D250763, AI768283, BG282957, AA548114, CB270753, BF515914,AW369265, AA312903, AI702437, BX471563, AA582936, AI907200, BQ215703,BM712460, AW504101, AI452675, BI459976, BE772886, AW576371, BM838528,AW401569, BE772887, AI218110, BE349982, BG723008, BM506721, AA592904,AI458250, AI272121, AW966715, AI572661, AI023105, BM831424, AI285500,AA808705, BU738082, AA506450XPF: BI522552, CB956135, BG620282, BG181154, CF529228, BQ013114,BX503907, BQ310815, BM671280, BQ011470, AW977575, BM710111, BG724387,AL705565, BE818393, AA291199, AA770518, BE837466, CD674166, AA774566,AI431784, AA256859, AW271424, AA638976, AV685090, BE818447, AA255461,AW242081, AA723776, AV692790, BE814005, AI653508, AA721794, BX103000,AA834535, AA292809, AA808363, BI459712, AA284141, AA639091DDB1: BM927667, BM545266, AL547974, BM559217, BM474381, BQ057079,BQ230722, BQ051604, BQ927173, BQ943701, BM799741, BU543084, BQ220481,BQ070702, BQ061047, AL521541, BU931018, BM469013, BU153954, BQ945468,BU165038, AL555048, BQ898580, BQ068618, BQ050859, BG764306, BQ057811,BU159948, BU508051, BQ065776, BM804642, BU845856, BM905933, BQ935651,BQ060859, BQ052770, BQ066316, BQ066118, AU125547, BM803322, BQ927550,BM552236, BM473607, AU121686, BG746666, BU535682, BM556709, BQ931953,BG831447, BG751027, BG677450, BQ683506, BQ642180, BM476800, BQ063089,AL549443, BM553844, BU178933, BI256821, BQ059091, CD652436, BQ962485,BU542556, BQ061252, BU156588, BQ642770, BU501977, BQ897254, BG762513,AU140587, BM046600, BQ070862, BI260236, BG747001, AU140248, BU154345,BQ060945, AU140289, BE743760, AU130230, BQ056360, BE794022, BG327224,BE747530, BI457215, AU140521, BM456004, BG481963, BG469259, BU943325,BQ943596, BQ213497, BM043469, BQ439584, AU140209, BG769813, CK000424,BQ279191, BQ438774, AU140418DDB2: BX401847, BX400795, BM460187, BQ688926, BM563807, BU159281,BX384437, BM008599, BE792938, BG479004, BQ940060, BE797218, BM560871,BX117885, BM018420, BM455897, BI256001, BX360369, BI868487, BX385303,BE799933, BM009575, BM553220, CA487463, BU174903, BX366945, BM850079,CD518332, BI915455, BG756997, BM782789, BI915534, BM554617, BG1318313,BE261143, BG913101, BE783395, AL566516, CB142981, BG035565, BX403426,BI838828, BG756904, BE885720, BX400794, BE018683, BI761524, BM746344,BX363265, AW247981, BM743623, BE536472, BE903342, AA309052, BG612441,BQ063694, BM744890, BX384436, BI255783, BM791178, BM924491, CD696204,BG106780, AL536826, BQ062583, BF974680, AW803143, BM783399, BF791778,BG759533, BX366944, BF974639, AL702736, AA278480, BG613246, BM840145,BM821166, AA311506, CD685541, BM920389, BF375336, BF576398, BX360368,BX384709, AL702729, BP431548, BI092193, BF382434, BQ001750, BI261116,BF184818, CF144601, AA128445, BE798846, AW802999, BF203187, BQ001644,BE247271, BQ003952, BQ575065XAB2: BX356659, AL518679, AL519886, BX424193, BM922374, BX381810,BX381811, AL557238, AL535342, AL525436, AL538943, AL518678, BX342559,AL565880, BX397557, BX383379, BX383380, BX382204, BQ053451, BX383814,BQ643137, AL561830, BQ050225, AL519590, CA488373, BU845857, AL560324,AL521338, AL534944, BX370726, AL560477, BQ935369, BU153161, BM476327,BG488778, AL516161, BX336874, BX392023, BM806807, BX364648, BI771923,BQ927835, BG488879, BM917523, BI822736, AL514374, BQ227702, BU541210,BQ221081, BM451911, BI772072, BX446010, BU855544, BU179797, BI823391,BM913999, BX448905, BQ054530, BG744221, BI910746, BQ956926, BG480524,BI261656, BM469671, BX464425, BI767323, BE253641, BI768994, BG386812,BQ063543, BI911058, BI518767, BE799838, BQ212885, BI459403, BU528530,BQ073165, BM048645, BE898732, BG468301, BE531308, BF311851, BI561194,BI560801, BF569424, BF686448, CD672626, BG425290, AL559075, BU170853,BE728374, BE871613, BE274104, BF314780, BE280310, BM456485, BU856266,BM020680, BI911907, AW837892UNG: BM926584, BM799989, BX438441, BX342506, BU165625, BU943452,BX378897, BM471137, BM928006, BQ228775, AL559968, BX379137, CF551970,BX378357, BX372231, BU161952, BU176423, BQ882986, BQ420300, BX361226,AV705903, BM919577, BX440252, BI823926, BQ048928, BM458045, BG105781,BQ962046, BU187892, CD643361, BX366166, BM541301, CD512104, CD686190,AU126319, BM718553, BE793197, BM449708, AI879177, BX429498, BE902908,BQ950839, BG326541, BG389571, BM012071, BQ670076, BF342799, AA290918,BG392330, BM799653, BX368057, BE734542, BG717638, BI198939, BI226322,CA454788, BG176725, BX347126, BE882172, BG256273, H09366, BI226401,AL079771, BE781768, BQ348703, BG390499, BF701762, BG176633, BE559523,BG282433, AU279887, BQ365552, BE883671, BE270595, BQ917973, BE258817,BE546123, AW401453, BG106747, BE268637, BI259016, BU168154, BX346912,T78215, BG481771, BX346928, CB130269, CB129289, BE261638, BQ348874,AA573859, BP429782, CD672939, BM751245, R25268, AA356048, BI766031,BM825376, BE263990, BQ322779MBD4: AL556619, BX372087, BM459663, BI767663, CD105484, BG032353,CD367008, BM690016, AL549313, BQ722669, BE561716, BI521142, BG032516,BI765468, BM465252, BI823689, BG686312, CA773665, BG716078, CD364595,CB989176, BF446103, BE622249, CA943572, BM314436, BI596708, AW964068,BI820928, BF033618, AW073379, BM857488, CA773226, AA741175, CA395073,CB136245, CA867841, BE541307, CB995679, AL578833, BM749974, AL553305,BE614377, CD557525, CB158348, AL553419, CB243592, BM462436, AU138081,AW138783, BU789775, AW193960, AA167425, AW195025, BG621850, BF509234,CA390195, BQ005967, BG613448, AW958704, AA939068, BF509053, AA167418,BU619480, BM476709, BM314740, BI462718, BM015493, BI544324, BF109031,AA905592, AA648364, BF109027, BF515981, AA167414, AI682256, AA011232,AI225045, BU153530, CA487592, BM857715, AA954283, BX390695, AW474165,AA825707, BI517400, BG742246, AA353798, CD678412, BG031116, BU732453,BM836637, BM709307, AI217321, AA247185, AA618259, BU678959, AW959666,CB144059, BI669635, AA171632TDG: BM479641, BM905541, BX370775, BM456725, AU128073, CD652973,CD657696, BG621267, AV708234, BE779060, BX331941, BX483618, BX382383,BF033788, BM476558, BX385505, AV649391, AV649244, AV649186, AA477864,BE181979, BX338343, CB145292, CD642813, CD644103, BM729260, BI760123,AL600750, BX509348, BX492385, BX476996, CD110168, CB994452, CB961815,CB267807, AL701375, BM915474, BG1314997, BF002914, AW590228, AW502250,AW136393, AL120270, AW051610, AI916834, AI868982, AI767246, AI669518,AI493141, AI360256, AI352697, AI332786, AA306938, AA257018, AA143198,AA131695, AA316331, CD109607, CD109351, CB989389, CB959951, AL699594,BF674842, BE502468, AI769788, BU566171, BX366012, AV654940, AL710869,BE784882, AI272154, BX340488, T34101, AW408102, BM457221, BF001989,AW138490, AW135094, H14409, BG940420, BU564446, AI869223, BE536675,BF241047, BE882613, AI435880, BF195990, BE080436, AW955279, CD050602,CA406412, AI272147, AI338205, BX646789, AA356499, BM556682, CB047650,CB047649, AI937774, AA360035NTH1: BM553336, AL536460, BM921161, AL566343, BM803928, BM472681,BG760523, BX438358, BM019398, BQ052788, BG821962, BI868261, AL545181,BI199103, BG766177, BI832477, BQ216562, BI757515, BQ431466, BQ052774,BE799423, BG519584, BG490410, BG747350, BG472059, BE746343, BI839163,BI196060, BG468596, CA488808, BF794685, BU182347, BG388749, BI226382,BE792632, BG248655, BQ881995, BF525435, BE797167, BM917598, BM451836,BF315844, BF312887, BG468607, BE790928, BG114969, BF337743, BE878633,BM677868, BF205470, AL545152, BQ958991, BG827031, BE266472, BE314558,BM811237, BF219845, BM831407, BX445525, BM424115, BM916696, BE313626,BG678320, BE262196, BF220231, BG331307, CB142490, BQ647925, BU622908,BQ575671, BF303821, BE891721, BE744813, BG468617, BU535559, BU634434,BM982354, BM129563, BE250955, BM821556, BM851724, BM725444, AW246140,BG469318, BX444894, AI610226, BX379864, CA488850, BE256091, AI818303,BU957532, BM129299, BM831443, BU902454, BF002443, AI968475, AW732463,AI424835, BU849457, AW103041NEIL2: BX401292, BX418848, BX386695, AL529804, AL530971, BX341864,BG759722, BQ942628, BG697467, BX427919, BI522685, BU158642, BX386994,BX333648, BI224185, AW411371, BE887573, BI113772, AL555248, BF305600,BI522781, BI520072, CB112109, BG700461, BM466367, BI601338, BU189576,CF145781, BM011284, CD558091, CF147070, BE736612, BE736412, CF139009,BF346473, AL524751, BF932051, CD671249, BF241652, BE153549, BI962581,BI793306, Z43722, BX340965, BI548326, Z46109, BU165567, BU193072,CB126915, BM729333, BM450046, BI789116, AI968247, BF761462, BF529055,BI793006, BU168329, BE153640, BI439197NEIL3: AL528028, BU170388, BX391576, BM458786, BU173488, BX112923,BQ687469, AU133212, BQ054308, BQ220911, BX348730, AL528027, BF700528,BG397668, BX391575, BE885006, BG026947, BF664630, BG758440, BF030084,BG495300, BF103925, BF217043, BQ441413, BE882646, BE865481, BF183915,BU689565, BG122779, BQ422423, T85431, AA373561, BG388415, AI307746,AA815079, CB145683, AU154583, AA677552APE2: BX325338, AL556617, AL519979, BQ277791, AL561128, BX395132,AL531548, BX433301, BQ050352, BM541964, AL559217, BX385097, AL528954,BI093915, BU931498, BM016132, BI859309, BQ921050, BI086544, AL582410,BQ428526, BE531337, BU553240, BM015733, BE737187, BI767790, BU184166,BU164122, CA495324, BE794688, CA495346, BI196397, BI912615, BX325337,BI669515, BQ229782, BM807966, BG709216, BX382166, BI333249, AL527692,BI909503, AL561839, BE616906, BM016478, BM726096, BE547006, BU942097,BE885110, BI837686, AL527693, BG024450, BE794496, BI915874, BX452361,BI911544, BG752268, BG386237, BF203315, BI858986, AL578831, BX394096,BI859870, BI256178, BF308964, BF310943, BF307805, BI223857, BI160978,BI160236, BE548576, BF981813, AL580878, BQ328004, BF931957, CB993692,BE280661, BI033411, AL711190, BE729174, CF146056, AL564091, BM682530,AL528953, BG438154, BX374269, BE265083, BX392349, CD366445, CD514757,BE795242, AI547012, BU540750, BE076387, AI547003, BG897167, AW386829,BI094006, AI907885, BF792036PARP1: BX425285, BM474368, BM473858, BM458759, BM458491, BQ216584,BM468375, BU164317, BM924278, BU149272, BM474022, BM905935, BG281447,AL542989, BQ946216, BQ918876, BU849139, BX438143, BX443246, BX420713,BU166033, BM012504, BM454330, BQ438889, BI091452, BX395490, BU844993,BM472955, BM555163, BX368206, BU171470, BM472260, BG280821, BX464445,BI833606, BQ882633, AU124412, BM463285, CK000195, CD653823, CF552559,AU124072, BE740909, AU131873, BQ213230, CK000758, BQ954235, BU156802,BX460089, BX450439, BI253230, BM450940, BM478605, BI334768, BQ222114,BM805846, CD651914, CD108986, BX462212, AU125041, BQ708310, BM043633,BM452637, BX388560, BM012492, CD521009, BF976506, BX431987, AU138067,BX421686, BM469381, BU942653, BM545681, AL517083, BG499313, BG533818,BU177793, BE270913, BU178223, BI908423, BE783663, BI087079, BE270845,BE899131, BE561235, BG177824, BG031594, BG393066, BE560200, CK000604,BE744678, BM799547, BM453457, BI093436, BG259918, BQ691997, BI113824,BG123019, AA401836, BM472073PNK: AL577752, AL542181, AL529432, AL578082, BX440754, AL529433,AL531397, AL563677, AL563673, BM552937, AL530233, AL555192, BM811617,BI523512, AL562615, BX385339, BM047190, AL582405, BI761490, BI765355,BG519795, BU957084, AL518252, BU541073, AL561123, BG912156, AL531398,AL529487, AL555795, BI488573, BQ943952, AL518253, AL555631, BI908075,BF971606, AL525997, BX331554, BU552518, BF314736, BI522840, BE312745,AL530223, BM011630, BI489474, BQ219713, BI766984, BI199796, AL519579,AL563456, BQ877856, BI834426, BG252407, BE799855, BQ055605, BQ067892,AL519578, BF315056, AL042657, BU956990, BI820868, BM045471, BM710277,BE394572, BG330783, BI909140, CB529741, BU527521, BM974879, BU619715,AV655619, BM917371, BI907002, AL526117, BU543115, BG118159, BE313034,BM982849, BI599430, CA425985, BM910694, BQ772660, BI770327, BE734945,BU622816, BG939419, AL525953, BI116338, BM923265, CF594119, BI822801,CA439970, BI827695, BE272050, BM819357, BE266096, BM687914, BE260690,AI984026, BX279591, AI830883Polb: AL572526, AL547658, BU157194, BG743462, BX383155, BU166001,BG251605, BI761008, BM790436, BQ430835, BQ434342, BG032291, AL705932,BI753835, BI915120, BI559405, AL558615, BX646755, BE394043, CD641318,BM928122, BX395185, CA314334, CD671591, CA313995, AU121247, AA916271,CD579745, BF131951, BQ188410, AI654868, CA439409, BI598628, AA130183,AL580395, BG506123, BU188474, CB125716, CB141008, AA172068, CB130159,AL702696, AA706903, BG025809, BU933432, AI827117, BM852849, BE280856,AL702481, AA172228, AA130175, BF693388, AI124907, AW957764, AW102789,AI825920, BM725613, BM678092, BX115761, BU934294, BF245489, AW070694,AI014834, BQ053443, BI461358, AW402160, AI689323, AA315716, BF219035,AI034273, AA856902, BM931013, BM684499, AA977230, BG339593, CB144384,AA809775, BQ574627, BM979958, BF590500, AW269977, AL120608, AI128173,AI057605, AI032461, AI017499, BF507649, AI087793, AW887363, AI949400,BG196141, AA723600, BG209074, BQ186692, BQ184899, BG212204, BE889161,CD110437, BG220062, BG200837MSH2: BM479882, AL528268, BX461033, BM557852, BM457765, BX431195,CD247876, BM543463, AU125214, AU125592, BQ878410, BQ225922, BI256610,CD655479, AU118136, BI090516, BU154902, BG756122, AU123630, CD656123,AU133361, CD519566, AU133333, BG773440, BI769598, BU178986, CB955666,AU131598, AU124367, AL563106, BX436686, BM475467, BI093054, AU120648,BU182908, BE792530, BQ223894, BI757481, AU131477, AU124664, CD656999,CD652376, CD657313, BG716960, BQ431632, BM834569, CD557029, CD693039,BE778241, BU934097, CD653693, BE779907, BG773429, BQ643544, BQ422633,BG388582, BG759539, BE894244, BG721947, BE268484, BE297145, BF033549,BQ221216, BI561347, BG773147, BE870260, AU123223, AA502616, AI792246,BX436685, AU126323, BF205395, AU144782, AU129482, AI823868, AU151529,BE254661, AU123102, BQ334510, BX413346, AW951649, BF834144, AU129404,BM788022, BM455023, CA843563, BU620631, AV693408, BU600314, AA287480,BE897216, AW402832, AA219060, AV732547, CB135442, AW003984, BE550379,BG499470, AW515731, CB143683PMS1: BX353664, BM479838, BX328949, BX327629, BM553209, BX117693,BM800196, BG193431, CB157373, BG168340, BQ429685, BX435290, BI464618,BG536475, BM723144, BF666456, BG163660, BG776017, BX452128, AL043809,AW945172, CB161021, BF699885, AL705101, CB131671, BG292439, BX419980,CB136057, CB268969, BG120880, BM677668, BQ771615, BF978494, BU599098,BM742128, BI545790, BG401461, CA415469, AI811371, CB852984, BG827501,BG719470, BG772717, BU623174, AL043785, AI458470, BE779274, AI636100,AA278390, BM820659, AI076038, BE350913, BG222592, AA781041, BX353663,BF056020, BG716188, AA833518, Z36291, BM009631, BU597828, BE350907,BG614286, BI438347, BM996496, AV730735, BE972698, CB144057, BF210947,AW269877, AA573397, AA393893, BQ574496, AA573406, AI660351, BF570703,BU854630, BG196186, AI367805, AA210907, AA393809, BG215472, BG207030,AA092955, AI277404, BG195153, AI278080, AA297925, BQ220187, BG182167,AW661801, BG209122, AV732009, AV731296, BP431712, AI828829, AI655707,AL600680, BU561560, AA282075PMS2: BQ951503, BM474394, BU153042, BQ881303, BU178449, BQ069438,AU140605, BG829980, BQ644101, BG720607, BQ436841, BU153051, AL708946,BU622416, BF568181, BU171754, BE884933, BQ221907, BM012037, BQ369274,BM701781, AL699728, AU280501, BE763779, BF840656, BG177103, BQ045117,BE304459, BX385541, BI548246, BM669686, BM144251, BX283912, BF674494,BU193656, BQ775383, AL701903, BQ232687, BI544939, BX283370, BM967072,BM148437, BE090126, BM967300, BI912345, BG565558, BU665027, BM147147,BX385540, BE090067, AL702122, BF114739, CB1111334, BG708195, AA151500,BI752285, BF697242, BU934799, BU728992, BF381825, BM698903, AV655809,BQ322673, BQ050630, BG776255, AA428236, BU604958, AI096500, AA256227,AI147872, AA256169, BM893955, BI037161, BF840055, AI539402, BM893782,BX095320, BQ221253, BE675175, BQ368913, BI829104, BE676031, CF124711,BU622696, BU620584, AI831722, BQ644699, AI341574, BM714146, BQ129192,BG398558, BF216419, AA297413, CB306237, AA707711, AA458667, AA206606,AA078218, AA418026, AW968473MLH3: AU121422, BQ878851, BQ716451, BG499557, BM551767, BU939998,BU183004, BM019183, CD103565, BX105329, CB118745, BM272299, Z78340,BM910096, BI752941, AV716905, AW340308, AW362032, BM738947, BE702562,AA679054, AA910059, CD701194, BM977366, AA766226, CD691614, BF540730,AI694991, AI792373, BG531828, BF207616, AA128984, H14680, BG119667,T08142, AV763342, AV764126, AA128983, BF364343, BQ002635, BQ008613,CA423918, BM792789, CA417441, CA418772, Z78339, W90440, N28386, D59901,R24851, BM684959, BM931907, AI685618, BG621929, AW007533, CA440408,AI768447, Z42933, AW385347, AI743250, AA649171, BF593505, AI683070,BQ316181, BM989024, AW896052, BM021521, AA682848, BQ428064, BM021241,AI768554, N99774, AI769320, BF197240, BF475901, BM023592, AW814434,BM146299, R20012, AL709475, BM023291, BF197600, AU147242, AA043269,AA043268, BM148801, CD678743, AI147056, N20033, AI825216, BF507953,AI934949, AI452776, W90107, AI857356, N71335, AA403079, N71385, AA249090Exonuclease1: AL561030, BX416336, BU159140, AU124774, BU166252,AL582335, AL517937, BU190454, BG762651, BG764476, BU179240, BU163048,BG120654, BE780022, BE260617, AA486526, AL043793, BE385439, BM788680,AL517936, AL530611, BG1111229, BM837022, BX280790, AV712138, BU616763,AL530610, CA419695, W79628, BF793400, AW390232, BE538507, BU178737,BX437336, BQ576087, BE082055, BM988542, CA446317, BQ776150, BM983504,BU617051, AW390243, BQ015148, BG251725, BU619316, BF435309, CD644038,AI693533, AW665143, BE464836, BF240758, BM479145, AL043794, AA489549,BM465399, BF478070, AA703000, AW977979, AI768937, AA485938, AU148568,BF979589, AA122095, AA122096, N74770, W79484, AI653837, AA972063,BE737930, AI968408, AA578654, AI859579, BE738387, BF870905, AI683464,AA812220, BU623456, CA430743, BE154204, BQ012304, BE274208, AI357911,AA485921, BF870910, AW663404, AI005418, AI023645, AA622919, BE085798,AW080921, AI040508, AI084836, AW664078, AA909643, AA832160, BI494500,BI494499, BG527833, BX470325, BF435984Poli: BX360120, BX370036, BX329075, BX436380, BM453196, BX370037,BM470558, BM450274, BG428228, BG532401, CD642641, BX378703, BG564733,BQ316794, BX360119, BM788353, BM714817, BG724156, BI560521, AA156839,BU958811, W60418, BG678675, BF219796, BG718281, CA431039, BI465231,BX378704, BE895738, BQ438265, AW852908, AW247603, CA418634, C18134,BQ086307, BM979833, BQ429462, BM918353, CA447232, BQ365259, CA446505,BU688625, C18844, BQ365051, CB243862, BU624232, BX386327, AW974469,AA878207, BQ102388, N57090, AA812734, AA856713, AA156602, AW418676,AW857684, R37923, AA648538, BM559425, AW150751, AA383550, AW880052,AW269829, AI634894, AW468818, BF111492, AA825419, AW070469, AA890447Rad51: AL541688, AL524788, AL530472, BQ053625, BM808716, BQ070341,AL526587, AL526399, BU182612, BX364160, BX349297, BQ438308, BU553012,BM910438, BM558629, AI347079, BM810067, BG826929, BG774318, BU508719,BI827729, BE262785, BU164989, AL563696, BQ278437, BE256767, BQ424388,BI826961, BF969466, BM804639, BQ918872, BU624430, BF311612, BF313593,AU118946, AL563755, BF970732, BG325079, AL524787, BM791694, BM018810,BG338607, AL530290, BE312219, BM011814, BM745228, BE883694, BG474115,BU931527, BF982698, AL526641, BG420079, AW732525, BF203788, BM557367,BG480368, BM972210, BE514829, BF684891, AU123935, BF313295, AL569030,AL526670, BE394150, BI197901, BQ070384, BG469700, AW006523, AA568782,AU145391, BG774691, AI419710, CF139164, BE280848, BI193363, BG470554,BM147841, BX475529, BX494877, BE890859, BU596395, BM555611, BM796675,AI670798, BQ224221, AW103435, AA873056, BE779265, BX475478, BE926933,BE186007, BQ316481, BQ316480, BF683589, BF764274, A018041, AW392597,AU100170, BM145097, BQ316451Rad51D: BX443779, BM915550, BU681257, AL559564, BI915527, BI254468,BG829674, BI823883, BM476837, BU521767, BQ961661, BI916871, BG475664,CD387861, AI692982, AA707504, BX327473, AL581158, BE382759, AL597240,BI915277, AW631291, N57184, BU786480, AU098391, AA352205, AA868613,BQ576251, AA868612, D59413, BU784751, BI561390, BX111725, BF905526,CA423187, BF905698, BM559794, BE827486, AW948521Xrcc2: BQ068576, BX283699, BQ430896, AW795339, BX490624, AL575509,CD365380, CD364971, CD299443, BX452455, CD103503, CB998612, CB241699,CB161449, CB160341, BX112725, CA436504, CA433495, BU681335, BU677265,BU508799, BQ771572, BQ229516, BQ217842, BQ030362, BQ028254, BQ026904,BQ017903, BQ000324, BM992214, BM710721, BM707110, BM353061, BM312475,BM054681, BM047586, BM023098, BI911022, BI907991, BI222447, BG621955,BG259639, BG121288, BF695702, BF573900, BF475941, BE962487, AV713355,BE768228, BE768138, BE716250, BE674520, BE617958, BE617517, BE243767,BE046093, BE044165, AW856234, AW795333, AW469111, AW338249, AW272847,AW192175, AW152595, AW081629, AI921359, AI866980, AI859056, AI812052,AI807730, AI761522, AI693828, AI636343, AI587436, AI469779, AI458271,AI423414, AI401226, AI357497, AI347767, AI346854, AI346825, AI343926,AI304763, AI304314, AI299263, AI223196, AI222728, AI220037, AI219853,AI140511, AI125306, AI094986, AI089590, AI051303, AI021980, AI018616,AA928999, AA782270, AL574032Rad54: BX403014, BX333113, BX363903, BQ277879, BU552753, BU932120,BX363304, BU553867, BX402970, BG679516, BU170678, BU173543, AL520104,BX414817, BU160045, BU932121, BQ225695, BX403013, AU124617, BG471582,BX363902, BI819429, BX363303, BG393630, BX386730, BU553362, BQ048878,BE797412, BG763599, BG258318, BF689785, BU855314, BM789908, BM720371,BE378872, BE397391, BE270285, BG768944, BM739336, BG030016, BX414818,BF304094, BM795109, BE742863, BU633988, BQ224151, BF308260, BM821481,BF663661, BX483240, BG323434, BF690175, BE614490, BX329613, BU634286,BG740710, CD696837, BE872543, CA414068, AI818766, BM754736, AI061463,AL040507, BG395239, BM773411, BM825772, AA313874, BG944997, BG827295,BU934008, BM772100, BF769132, AW516286, BM745768, BM765732, BG720800,AU148441, BM746228, BM678712, AA227600, AW236802, AA582917, BG114710,BM753165, AI990748, BM753679, BM745825, AA724587, BU849113, BX106292,AL520105, BI255111, BE613982, AI372035, BM857179, BF913956, BE708827,AA227900, BM801845, AW003486BRCA1: BQ679749, BQ068830, BU194336, BU155689, AU122476, BM452288,BQ683955, BU552955, BU163307, AU142729, BQ878445, BU171200, BG681276,BQ676829, BU163141, BQ681242, CF121736, BQ422380, AU125312, BF508987,BF791668, BM042892, BU147444, BQ677666, BQ215100, CB155501, BG178466,BF983078, BG777447, AI992040, AL704228, AI589028, BF794879, BG257190,AL135363, AA608570, BG530796, BM800251, BM042282, CB118225, BE264293,AW295197, AW968546, AA205436, BE043993, AW968720, AI915085, CB158976,AA804632, AW504244, AU148997, BE018878, BE206562, AA702344, BQ214737,AW514868, AA812019, BE564528, AI684595, BU617173, AA486004, BU679389,BX102233, CB150491, AA814998, AA484941, CF142324, CF138586, CB136844,CB108172, AA773331, AI680547, BU677011, BM755214, CF596982, CF143993,AA111870, CF144118, A040685, BM988066, BF447679, BF028959, BQ308670,BX644276, H90415, AW673569, BM755305, AA086435, BF795489, AW408596,BQ378479, AW575729, BQ378695, AW964452, BE560149, BX497486, AI217721,AL043576, AA205474, AA917008Ku80: AL542654, AL537322, AL556485, AL552210, BM927751, AL557736,BX418952, BM801948, AL541582, AL516832, BX425176, BM905671, BX458362,BU193782, AL540737, BQ679008, AL550730, BM471778, BX446693, BU146999,BM560171, AL548391, BQ213485, BQ220595, AL542266, BU542616, BQ651471,AL541946, BM803480, BX439845, BQ899268, BQ672013, BM800555, BX419500,BX415043, BM543372, AU131739, BQ679661, BU188691, BQ059109, BM451420,BQ880232, BQ645929, BM467250, BM453905, BQ650072, AU142506, BU188386,BM451883, AU124221, BU168862, BQ649861, BU170704, BU155107, BU146251,BQ226372, AU125704, BU177113, CF552735, BX439846, BQ424597, BM549064,BU509423, BM809313, BI259161, BQ878391, BU158765, BQ226911, BX421454,AU119267, BQ898623, BQ425827, BG827810, BQ642849, BU177743, CD107663,CF265072, BM466124, AU141293, CD243745, AL575698, BQ921196, BQ427943,BM470728, AU131108, BG576028, AU131971, BU150184, AU132099, BU176974,BQ223936, BX447056, BU153309, BM466876, AU132244, AU122435, BQ229983,BQ881903, BM454471, BQ924231XRCC4: BX362079, BX340876, AL543920, BM471375, AL551668, BI760531,AL558342, BI770803, CD580212, BG682493, BI758185, BG699970, BG505339,BI822602, BI463813, BG772422, BM465969, BU664243, BG776379, CA394395,BI464058, BQ226357, BI828556, BQ233170, BF183927, CB962180, BE748849,BE748380, CB146884, BF211589, BX281210, BQ421318, AW950192, BF572503,BM846671, AA314379, BF669890, CD706606, AL580186, AA447878, BX279574,AL575167, AA258143, CB144620, BG500252, BF247013, BG499117, AA448976,BF107431, BF214359, R19860, AV717223, CA453949, BG497598, AA065267,BE254850, BU171074, BM564730, AU099389, BE781259, BE780721, BE778165,AA398779, BE781955, BE783342, AA398935, AV743689, AL570210, BF895164,BF242563, BX362078, R14027, CD358588, AI795996, BG282107, BG206341,BG204714, BG199056, BG193494, BG219448, BG218328, BX389897, BG221405,BG208423, BG204713, BG201112, BG196512, BG187196, BG192946, BG214239,BG211030, BG208424, BG203200, BG198531, BG198530, BG186701, BG186093,BG181983, BG213659, BG214740Tin2: BM911894, BQ941808, BX398174, BM915062, BQ066985, BX430064,BX347075, BX387627, BI837194, BQ423479, BX347045, BI871294, BE747943,BX429614, BX347087, BX337436, BU942629, CD244144, BX388585, BF793349,BX346850, BX388520, BX423719, BX367761, BG420146, BE903807, BE727299,BX388767, BX428959, BX430065, BX326045, BX346831, BX355414, BX428954,BX367991, BI193188, BX388709, BX398173, BX394341, BX442338, BX326233,BX432669, BQ707785, CK005692, BE562849, BU187043, BX429889, BM541314,BX444001, BX474320, BX386120, BX355792, BX395024, BQ218393, BE408455,BX381411, BX423718, BQ222471, BF125394, BE410701, BX388630, BI909268,BX374920, BX375500, BX368189, BF125791, BM549897, BF125418, BE743717,BX333804, BX450403, BI754471, BI760932, BX388787, BX356844, BI518422,BI488522, BQ707493, AA428113, AV686147, BQ720769, BM545840, BE383960,BI764031, BI767028, CB216205, BG824273, AW402903, BI835774, BQ707958,BM452819, BI833476, BI755739, BM919172, BX326360, AV693747, BI116486,BX368042, BE882159, BI821458Sir2: BM544569, BX445007, BI834120, BM547962, BQ228980, BU507144,AL550142, BX367337, BQ072979, BI766740, CD624348, BQ052789, BU182713,BQ068262, BM920249, BQ951302, BE379525, BI862361, BX375262, CF264878,BF528797, BM462565, BX340941, BM806242, BI518634, CD515474, BX453795,BU195684, BQ068338, BQ058696, BE798693, BQ645221, BG437042, CD517619,BX380923, BQ068347, AL519386, BU197397, BM903578, BI554088, CD624352,BQ221442, BM473470, AL533183, BM924936, BI766260, BI918160, BI762157,BF034485, CD674710, BQ653076, BI603360, BG723057, BG339784, BM546244,BQ929517, CD624347, BI523850, BX428185, BG339736, BG468891, BG386360,BI838558, BI771058, CD624353, BF345522, BI823957, CD558177, BI766390,BI768954, BI768415, AL549311, BI907256, BM906233, BG819884, BI910251,BI760600, BI517372, BM805816, BF529638, AL561653, BQ430510, BQ339694,CB150996, BF975840, BG032959, BF531032, BI756237, BF975705, BU196170,BI763858, BI918541, BI524122, BG332544, BG328012, CD624351, BF686436,BG288542, BI838925, BE867361MGMT: BU931774, BU859113, BU172662, BQ641434, BQ220709, BI771279,BI520278, BG753063, BU850242, BQ228817, BQ710379, BI520938, BG764104,BX094941, BQ279107, BI226276, BU858086, BX509195, BM759902, BI520029,BU154192, AL520114, BM974121, CF130478, BM738844, CB992752, BI772512,BG436862, AL520115, BM973348, BG249568, BI520980, AL524961, CB993639,BM009017, BU845865, BM970224, BX373012, BU616455, CB055208, BI225271,BI333401, BX376972, CB055209, BU947266, CB997161, BE858532, BM972582,AI719186, BM670373, BU845870, AA978354, BM744653, BG340352, BQ222473,BM758658, CD369999, BG183775, BX351398, BE541556, CD249663, BM744647,BU737340, BM754382, BG1381704, AW168149, BM712082, AI963126, AW274265,BF109578, BE464809, BM711175, AI016474, AA126722, AI143841, AA948354,BU786059, BM049297, AA779559, AI052155, W58681, N95214, AA988766,BU566480, BX349121, BQ072274, AI057145, BM823702, AI040746, W25247,AA677158, AA136191, BX383619, AI123988, R72558, BQ217761, AW804292,AA565025, BE774145, AA868690DUT: AL576853, AL519489, BM757904, CD247125, BM457507, BI091680,BQ440183, BM915011, BM740990, BU600705, BE386365, AL532465, AI686520,BM475441, AW968574, BF338018, BF206146, AI680930, BG682494, BI836025,BE221492, BI255334, AI951891, BI868234, AW968748, BM470935, BG700386,BE897174, CB529208, BU677683, BU677665, BU620392, AL554011, BQ777742,BE254729, CA777885, BU597092, AU119115, BU623296, BG717317, BG655751,AW162006, BI670458, BG705392, BE902236, BG717215, BG53201, BG610639,BG677850, AI635074, AI261871, BI091131, BG163981, BG113287, BE706306,AA056738, BE644721, CB110414, BF317403, BE551158, AA737006, BM554499,BE222283, AL532464, BM840182, AA256721, A373097, AW629827, CA778151,CB117412, BE549576, BG505144, BE218639, BF058963, AW341118, AW967946,AA278799, BE504213, AI191219, BI860728, AW962792, AI697600, CB137303,CB133275, BE502892, AA291243, AI199667, AA446533, BE673841, AI937879,AA434589, AI986329, AA433910, CA488337, BF938984, BU608498, BM559498,BU683317, BG403290, BM817453TIMELESS: BM467715, BM927658, BM541298, BM801216, BQ052552, BQ945096,AL560919, BQ068552, BU845242, BQ071352, BU930918, BU854737, BQ068451,BQ961203, BQ055183, BU500665, BX390921, BX401304, BQ962781, BQ672871,BG749383, BX346012, BU521442, CF242984, BU552412, CD653932, AU125640,BU54348.5, BQ927368, BQ051381, BI222498, BG822789, BQ944034, BM046877,BU146750, BU956003, BQ670516, BQ061549, BG757741, BE797452, CA430803,BG819936, BE746308, BU187951, BE794062, BM013386, BE795708, BU553769,BM552373, BQ424129, CD654639, BM910771, BG388233, BE729276, BM013167,BE791318, BE514198, BM740568, BX350660, BG110568, BX110927, BI087328,BQ958679, BE727460, BG823400, BE729002, BG289919, BF971197, BM048813,BE514731, BE314800, BQ887260, BE745259, BE408808, BE208475, CA489086,BG478136, BU188642, CD673319, BE389356, AW382754, BE793649, BQ214512,AW383633, BM793905, BM789297, BQ221649, AW383534, AW673493, BM018763,CB990372, AW383548, BQ937242, BQ678339, BU172010, BU167922, BU163559,BQ679317, BQ679254, BQ679251Pif1: AI655645.1, AI280491.1, AI654749.1, AI333976.1, AA743647.1,AA872541.1, AA279102.1, AA278838.1, AI827264.1, AI652391.1, AW004048.1,CN358868.1, CN358870.1, T85126.1, T88870.1, CN358869.1, T54683.1,T54599.1, CN358871.1, CN265097.1, AA464521.1, BG231673.1, AA642924.1,BX109827.1, AI696210.1, AI745642.1, AI984536.1, AW170361.1, AW590310.1,AW663962.1, AW801494.1, BF516453.1, BG951026.1, BI116535.1, BM043514.1,W60651.1, W60880.1, BM888249.1, BQ061682.1, BQ065272.1, BQ958807.1,BU502486.1, CA310274.1, AA464522.1, BE280562.1, BF316643.1, AA827755.1,CN259034.1, CN277199.1, AA973831.1, CN259037.1, CN259036.1, BX283578.1,AI889087.1, BG323851.1, BI063248.1, BI063942.1, BQ315498.1, BE148134.1Mms4: BI918962.1, BE613887.1, AW672839.1, R86709.1, R85191.1, H80469.1,CN290252.1, CN281043.1, CN342824.1, R83093.1, BX333338.2, BX327691.2,D25658.1, AW955836.1, BE378681.1, BE542457.1, CN484982.1, R87430.1,BE887145.1, BE895828.1, BE897152.1, AU118079.1, BF244395.1, BE547467.1,BG028794.1, BG000465.1, BG167025.1, BG167838.1, BG255030.1, BG256327.1,BG436043.1, BG393362.1, BG498016.1, BI084419.1, BI088431.1, BF793987.1,BG996952.1, BI255051.1, BI256195.1, BI258650.1, BM462968.1, BM800592.1,BM802118.1, BM809503.1, BM906256.1, BM918347.1, BQ072173.1, BG992888.1,BQ216341.1, BQ218372.1, BQ230208.1, BQ232225.1, BG116791.1, BG120099.1,AA243757.1, AA443229.1, AA481119.1, AA774064.1, BX108233.1, BX333337.2,BX443561.2, BX328975.1, AI305107.1, BP429202.1, BX509606.1, BP430970.1,AW250729.1, BP430731.1, CK023740.1, CN482590.1, BQ082456.1TopoisomeraseIIIa: BM462184.1, BQ226006.1, BQ215187.1, AI933546.1,AI694682.1, AW005757.1, AI871758.1, AI627306.1, AI357363.1, CN431712.1,CN431714.1, AI131044.1, CN431713.1, CN431717.1, CN431715.1, AI863107.1,N21546.1, AI652693.1, AI637907.1, AI917456.1, AW370762.1, AW375839.1,AW411282.1, AW411283.1, AW449041.1, AW449710.1, AW450373.1, CK300631.1,CD619024.1, CD619023.1, AW513442.1, AW748535.1, AW954652.1, BE062796.1,BE062799.1, BE062870.1, BE062878.1, BE275290.1, BE294390.1, BE297536.1,BE383990.1, BE384385.1, BE388003.1, BE389807.1, BE408865.1, BE410098.1,BE886538.1, AU125151.1, AU130137.1, BF307775.1, BF345890.1, AW601235.1,BF060985.1, AU148874.1, AU152155.1, BF772443.1, BF913078.1, AL555800.3,AL578214.3, BG281391.1, BG333658.1, BM046431.1, BM046548.1, BM049492.1,BM553540.1, BM683614.1, BM804018.1, BM817686.1, BM910465.1, BQ187307.1,BQ334676.1, BQ342906.1, BQ673046.1, BM929666.1, BQ883495.1, BQ897944.1,BQ929795.1, BU159208.1, AL601602.1, BU542042.1, BU191611.1, BU902730.1,AA307047.1, AA325934.1, AI206124.1, AI206134.1, CB123137.1, R45840.1,AL040785.1, BX394136.2, BX359327.2, BX359328.2, BX348607.2, AI969044.1,AI978571.1, BX499775.1, AW270386.1, AW351792.1, AW370749.1, AW370750.1,AW370754.1, AW370756.1, AW370757.1, AW370758.1, BU169620.1Mus81: BI828324.1, BI822910.1, BI772783.1, BI766615.1, BI551731.1,BE313033.1, BF317447.1, BG912942.1, BG388554.1, BG336401.1, BM926996.1,BG334598.1, BG330488.1, BG328798.1, BG327203.1, BG165822.1, BM561952.1,BM795493.1, BI871701.1, BI909656.1, W05036.1, BM787599.1, BM015068.1,BM762869.1, BM715429.1, W46505.1, BM051590.1, BM193717.1, BM820646.1,BM673914.1, W46441.1, W46397.1, W46466.1, D81040.1, D81583.1, N66260.1,BI083900.1, BI084569.1, BI058696.1, N74665.1, BI015411.1, BI225125.1,BI261175.1, BI334628.1, BI520226.1, BI520852.1, BI495735.1, BI495736.1,N99229.1, BI818186.1, BI818415.1, BI821127.1, BI816797.1, AA278513.1,CA426286.1, CA428478.1, CA439511.1, CA488369.1, AA310067.1, AA321039.1,AA325265.1, AA353351.1, AA361208.1, AA361844.1, AA410784.1, AA412362.1,AA425903.1, AA483867.1, T24587.1, AA588568.1, AA742229.1, AA742315.1,R07365.1, AA767217.1, AA808486.1, AA811878.1, AA830456.1, AA831614.1,AA935774.1, BM828633.1, W86124.1, BQ004754.1, BQ007168.1, W94929.1,W92200.1, W96213.1, W96307.1, BQ219750.1, BQ421044.1, BQ673374.1,BQ716313.1, BM147893.1, BQ951454.1, BQ787518.1, BU626130.1, BU630449.1,AA195097.1, AA195293.1, AA235399.1, BU855063.1, AA256727.1, AA258031.1,AA261839.1, AA262485.1, AA262698.1, CB989437.1, CB995814.1, BX329339.2,BX363788.2, BX363789.2, BX364239.2, BX364240.2, BX376440.2, BX376441.2,BX352736.2, BX352737.2, BX400774.2, BX393197.2, AW008546.1, Z46145.1,Z41778.1, AW080590.1, AW081005.1, CF130974.1, AW057824.1, AI174987.1,CF272441.1, AW292451.1, AW296475.1, AA989261.1, AI004770.1, AI027750.1,AI078127.1, AI161039.1, BX117949.1, AI223161.1, AI198223.1, AI289132.1,AI291347.1, AI312012.1, AI423724.1, AI355186.1, AI444946.1, AI565701.1,AI568723.1, AI401565.1, AI589837.1, AI561084.1, AI696162.1, AI701699.1,AI708157.1, AI796616.1, AI809463.1, BE843407.1, AV683439.1, AV684455.1,AV692941.1, AV695347.1, BE871764.1, AV706138.1, AV737334.1, AV749817.1,D53819.1, BF337818.1, BF340725.1, BF345411.1, AA836608.1, H70568.1,BF594105.1, BF594519.1, BF742954.1, BF801584.1, BG026984.1, BF940759.1,AL535449.3, AL535450.3, BG393857.1, AW387757.1, CF995326.1, AW469599.1,CD632462.1, CD632461.1, AW905089.1, AW954207.1, CN336481.1, CN336479.1,CN336477.1, CN336478.1, CN336480.1, AW967947.1, CN422010.1, CN422011.1,CN422012.1, AW978460.1, CV028339.1, BE328203.1, H25803.1, BE379473.1,BE396264.1, BE396772.1, BE513701.1BE564111.1, AW365100.1, AW365101.1SIRT1 (Sirtuin): AI037953.1, AA236993.1, N23557.1, H98832.1, R86123.1,AA044634.1, AI381553.1, AA608812.1, AI378978.1, AI367389.1, CN357085.1,AI972705.1, H12698.1, AI217748.1, CN357086.1, BE072031.1, BE081871.1,BE245026.1, BE463430.1, AV660110.1, AV660133.1, D59300.1, BE883278.1,D62968.1, AV704288.1, AV704956.1, AV750129.1, BF445130.1, BF692058.1,BF848464.1, BF848494.1, AW615289.1, AV718812.1, AV720195.1, BF590111.1,BF796692.1, BG026102.1, BG036612.1, BF999696.1, BG178600.1, BG282746.1,BG283059.1, BG496097.1, BG498089.1, N68314.1, BI091351.1, BG705339.1,BG717615.1, BI258271.1, AL599794.1, BI520244.1, BI869083.1, BI918557.1,BM273130.1, BM475115.1, BM697223.1, BM905888.1, BM980158.1, BM986798.1,BQ025488.1, BQ219206.1, BQ226337.1, BQ631955.1, BQ632248.1, BM152225.1,BU186744.1, BM452557.1, AA251252.1, AA382573.1, AA452304.1, AA460952.1,AA461259.1, AA828109.1, BX105044.1, AI751813.1, AI751814.1, AI807525.1,AL042303.1, CD110682.1, AW007728.1, AW020605.2, AW021852.1, BX505161.1,AW504399.1, CK820052.1, CK820053.1, AW967429.1, AW996552.1, BU935054.1Esp1: BM456594.1, BM051112.1, BM049903.1, BM044277.1, BM013405.1,BI262337.1, BI117483.1, BI200147.1, BI195989.1, BI023133.1, BG821987.1,BG767950.1, BG760762.1, BG756617.1, BG684814.1, BG493667.1, BG490228.1,BG480927.1, BG479609.1, BG469946.1, BG386315.1, BG337498.1, BG328178.1,BF932278.1, BF973206.1, BF972380.1, BF764335.1, BF742768.1, AA780037.1,AA581005.1, AA580948.1, AA548572.1, T86767.1, AA455415.1, T86675.1,AA339975.1, AA248889.1, BU856483.1, BU855930.1, BU844826.1, BQ958098.1,BQ939849.1, BQ894059.1, BQ893225.1, BQ882493.1, BQ881349.1, BF924624.1,BQ361233.1, BQ069829.1, BQ052875.1, BQ052507.1, BQ014621.1, BM905149.1,BM837056.1, BM797577.1, BM468940.1, AW207246.1, CF137736.1, CF137709.1,CF137594.1, CF135495.1, BX483646.1, CD579284.2, AW009863.1, AW008862.1,CD359905.1, CD299902.1, BX415621.2, A816969.1, AL046060.1, A800823.1,R42883.1, AI458447.1, AI446360.1, AI283098.1, AI268609.1, AI214569.1,AI127437.1, R21501.1, AA928961.1, AI023991.1, AI023899.1, AI022797.1,AA948058.1, BF512245.1, BF698487.1, BF697145.1, BF686746.1, BF683733.1,AA694341.1, BF314077.1, BF155328.1, BF154952.1, BF154938.1, BE538398.1,BE467107.1, CR746800.1, BE019694.1, CN356489.1, CN356487.1, CN356486.1,CN356485.1, CN356484.1, CN356488.1CN356495.1, CN356494.1, CN356493.1,CN356492.1, CN356491.1, CN356490.1, AW867242.1, AW497592.1MPG: BE567173.1, BF678850.1, BG769688.1, BM663214.1, BM690361.1,BM707428.1, BM750136.1, BM771938.1BM796074.1, BM796301.1, BM822805.1,BM823382.1, BM823606.1, BM825519.1, BM831379.1, W69334.1, W69335.1,BM852297.1, BM909194.1, BM914408.1, W76127.1, BM922561.1, BM970297.1,BQ053339.1, BQ224541.1, AA010929.1, AA011317.1, BQ416588.1, BQ416589.1,BQ416891.1, BQ416892.1, BQ430971.1, BF174738.1, BG469815.1, BG479289.1,BG574541.1, BG686214.1, BG779735.1, BG746848.1, BG763639.1, BG823448.1,BG830627.1, BI253826.1, BI259059.1, BI524092.1, N90880.1, N91934.1,BI552950.1, BI713520.1, BI759543.1, BI818264.1, BI820665.1, BI791744.1,BI870400.1, BI858760.1, BI964886.1, BI906325.1, W17097.1, BM194361.1,BM471029.1, BM543869.1, BM552363.1, BM556912.1, BU565588.1, BU631253.1,AA187311.1, AA187412.1, BU684880.1, BU687924.1, BU858254.1, BU860310.1,BU956115.1, CA337274.1, CA423548.1, CA438664.1, CA441991.1, CA488184.1,CA488371.1, AA299077.1, AA285256.1, AA491244.1, AA503832.1, AA527886.1,AA568795.1, AA578450.1, AA603076.1, AA621361.1, AA732078.1, AA761676.1,AA767201.1, AA768478.1, AA768552.1, AA806008.1, AA853981.1, AA857130.1,AA026824.1, AA026957.1, BQ447759.1, BQ668747.1, BQ675987.1, BQ676108.1,BQ676179.1, BQ676405.1, BQ678708.1, BQ678722.1, BQ680099.1, BQ682489.1,BQ684616.1, BQ686780.1, BQ773643.1, BM147473.1, BM149314.1, BQ888825.1,BQ921148.1, BQ927180.1, BQ945550.1, AA065084.1, AA064997.1, BU195384.1,BU149567.1, BU157028.1, BU168113.1, BU172290.1, AA113980.1, AA113972.1,AA115941.1, AA122238.1, AI695611.1, AI739369.1, AI750356.1, AI754347.1,AI765874.1, AI798386.1, CB267218.1, BX281276.1, AI817691.1, AI913581.1,AI922106.1, AI928586.1, CB997674.1, BX395164.2, CD105357.1, BX353976.2,BX372753.2, BX392614.1, BX357530.2, BX391673.2, AI961117.1, CD368856.1,AW007326.1, AW072347.1, AW083039.1, AW131829.1, AW149104.1, AJ573841.1,AJ573842.1, AW204546.1, AW206735.1, AA971357.1, AI015443.1, AI037999.1,AI089498.1, AI131317.1, AI143697.1, CB044049.1, CB044050.1, CB048492.1,CB048493.1, CB048779.1, CB049248.1, CB049249.1, AI149080.1, AI188471.1,AI205596.1, AI208926.1, AI209171.1, CB128954.1, AI203215.1, AI345964.1,AI350908.1, AI382391.1, AI453105.1, AI457314.1, AI521404.1, AI553664.1,AI569449.1, AI580320.1, AI679185.1, AI695608.1, BF001487.1, BF002356.1,T28409.1, BF109116.1, BF219424.1, AA862053.1, BF664362.1, BF669361.1,BF526924.1, BF572325.1, BF790595.1, BG033001.1, BG033146.1, BF974328.1,BF940863.1, BG165742.1, BG153198.1, AL520051.3, AL520052.3, AL521514.3,AL521515.3, N26769.1, AL524386.3, AL524387.3, AL527543.3, AL527544.3,AL530961.3, AL530962.3, N30855.1, BG284431.1, BG430698.1, F04542.1,AW291955.1, CF993786.1, AW452798.1, AW590115.1, AW593038.1, CK902061.1,CN266626.1, CN266627.1, CN266628.1, CN266629.1, CN266630.1, CN266631.1,CN266632.1, AW973756.1, BE045501.1, BE207420.1, BE275484.1, BE385924.1,BE394756.1, BE408565.1, BE465105.1, BE502294.1, BE538276.1, BE733520.1,BE735753.1, BE780181.1, AV694099.1, BE908188.1, BE909470.1, BF000140.1,BF000556.1, CB114652.1, CN276703.1, N41382.1, BQ694950.1Poll: BQ052050.1, BQ072779.1, BQ232853.1, BG823706.1, BE264721.1,BI758654.1, BE539840.1, BE541711.1, BG389885.1, BG331116.1, BI770175.1,BE880092.1, BM544877.1, BI825514.1, BE974036.1, BI827122.1, BI910222.1,BI906881.1, BI909106.1, BI906886.1, BI772047.1, BG945087.1, BI091406.1,BG770791.1, BG764790.1, BG763955.1, BG751184.1, BG749688.1, BG490202.1,BF171635.1, AL562678.3, AL541662.3, AL526446.3, BG149290.1, BG024414.1,BF807384.1, BF591938.1, BF476040.1, BF475279.1, AA807380.1, AA742404.1,T81701.1, CA406519.1, CA405484.1, BU855318.1, AA234405.1, BU618135.1,BU194042.1, BU184350.1, BU191914.1, BU159682.1, BQ957310.1, BQ932866.1,BM148925.1, M145476.1, BG118565.1, BG056482.1, BQ311579.1, BQ276405.1,AL702021.1, AL702011.1, AL698041.1, M923533.1, BM908370.1, W69888.1,BM824844.1, BM801377.1, BM354225.1, BM273229.1, BI912822.1, BI909369.1,AW406239.1, AW418802.1, AW377370.1, AW377335.1, AW377300.1, AW377298.1,AW377264.1, AW377257.1, AW367852.1, CF147037.1, CF140209.1, CF140207.1,BX426881.2, BX372137.1, BX384565.2, BX384564.2, AA991853.1, AA927738.1,AA922738.1, C A976110.1, CB151800.1, CB131141.1, AI538103.1, AI922500.1,BX333693.2, BX351220.1, BX361517.2, BX382774.2, BX398077.2, BX398078.2,BX400678.2, BX374468.2, BX390819.1, BE144586.1, BE162920.1, BP871239.1,CV025896.1, BE392666.1, BE562674.1, BE744935.1, BE819364.1, BE905104.1,BE938379.1, BE938389.1, AV705731.1, AU118033.1, AU122737.1, AU141478.1,AU144697.1, BF433511.1, AW673654.1, AW731648.1, AW752914.1, AW752922.1,AW768836.1, AW769721.1, CK820138.1, CK820139.1, CN304569.1, CN304570.1,CN304571.1, CN304572.1, CN304573.1, CN304574.1, CN304575.1, CN304576.1,H11886.1, CD243848.1, BX448084.2, BG488800.1, BI027900.1, BI460283.1,BM479907.1, BM541632.1, BM563437.1, BQ232479.1, BQ923975.1, BQ954336.1,BU195355.1Polm: BI914097.1, BI908863.1, BI907196.1, BI858799.1, BG700584.1,BM920340.1, BI769194.1, BG483069.1, BG499408.1, BG910514.1, BI029339.1,BI001584.1, BI752559.1, BI838830.1, BI857029.1, BM739081.1, BM739884.1,BM739966.1, BM744174.1, AV692664.1, AV697631.1, AV697781.1, AV699741.1,BF102699.1, AU129189.1, AA694217.1, AA766124.1, AW613566.1, BF530871.1,BF819685.1, BF895261.1, BG253328.1, AL523598.3, AL529987.2, AL529988.3,AL562232.3, BG231904.1, BG386713.1, BM148157.1, BM148944.1, BM149019.1,BM151641.1, BU517199.1, BU520948.1, AA077726.1, CA388951.1, CA487306.1,CA487434.1, AA298793.1, CA944555.1, AA507121.1, AA605050.1, AA769270.1,AA814924.1, AA815032.1, AA832314.1, AA917987.1, BM744180.1, BM747399.1,BM752457.1, BM755685.1, BM756419.1, BM756478.1, BM804478.1, BM808078.1,BM853841.1, BM911482.1, BM911832.1, AL700896.1, BQ015336.1, BQ214851.1,BQ686436.1, AA046576.1, AA046663.1, BG1316876.1, BM147833.1, BX390956.2,BX350666.1, BX385063.2, BX385064.2, CD109226.1, BX354131.1, BX354132.2,BX351215.2, BX376517.2, BX376518.2, BX395027.2, BX335337.2, BX335338.2,BX383403.1, BX383404.2, BX383221.2, BX402828.2, BX402829.2, BX384388.2,AI025113.1, AA928729.1, AI057140.1, AI208523.1, AA910584.1, AI365238.1,AI365240.1, AI392687.1, AI419960.1, AI458707.1, AI459543.1, AI469103.1,AI627311.1, AI652512.1, AI654109.1, AI738949.1, AI767982.1, AI638032.1,CB990757.1, BE241524.1, BE241656.1, BE241950.1, BE242681.1, BE242829.1,BE242932.1, BE243489.1, BE244287.1, BE247085.1, BE247483.1, CR735361.1,BE503442.1, BE789823.1, AV685517.1, AV688018.1, AV688777.1, AV689313.1,AV689923.1, AV691130.1, BX393262.2, BX435165.2, BX419627.2, CD252488.1,BX477278.1, AW070683.1, AW137352.1, AW182301.1, CF529830.1, AW207742.1,AW296010.1, AW361738.1, AW402473.1, AW575118.1, AW578625.1, AW592532.1,CN480773.1, CN304577.1, CN304578.1, CB104684.1, AI438940.1, CB266611.1,CN293022.1, AA078383.1EndoV: BQ279208.1BM554738.1, BM911430.1, BI830073.1, BI829389.1,BG681481.1, BG819544.1, BI117355.1, BI551407.1, BQ287914.1, BQ050806.1,BQ024655.1, BQ021027.1, W87446.1, BM926784.1, BM128679.1, BM128571.1,BM128352.1, AA720808.1, AA452047.1, AA363057.1, AA351281.1, BU621053.1,BU553848.1, BU527982.1, BQ957094.1, AA056490.1, BQ923151.1, BQ923003.1,BQ772531.1, BQ435901.1, BQ429806.1, BQ429804.1, BQ427432.1, BQ417371.1,BQ342726.1, R14533.1, CB143295.1, AI370900.1, BX334744.2, BX355957.2,Z41683.1, AW157406.1, CK819308.1, CN288502.1, CN310874.1, CN310875.1,CN310876.1, AW975617.1, H09974.1, BE619534.1, BE905153.1, BE938460.1,BF686490.1, BF477753.1, AL541721.3, AL569045.3, BG749082.1, BI549266.1,BI550542.1, BI550562.1, BI837982.1, BI917934.1, BM007984.1, BM008131.1,BM043073.1, BM047737.1, BX092874.1, AI219827.1, AI249283.1, AA926664.1,AI223657.1, AI290823.1, AI300416.1, AI311040.1, AI349370.1, AI393803.1,AI307620.1, AI571214.1, AI635870.1, AI660440.1, AI681370.1, AI702794.1,AW001931.1, AW136860.1, AW204051.1, AW438764.1, BE041666.1, BE043347.1,BE049112.1, BE219425.1, BE219749.1, BE672512.1, BF194919.1, BF196166.1,BF718311.1, BF840896.1, BM662670.1, BM665527.1, BM671155.1, BM684408.1,BM689003.1, BM710007.1, BM711618.1, BM930887.1, AA451847.1, AA593804.1,AA617805.1, T96971.1KNTC2 (NDC80): BG612856.1, BG532554.1, BG531886.1, BI093871.1,BG500282.1, BI825656.1, BE561023.1, BE564344.1, BE565202.1, BE566742.1,BE903220.1, BQ233581.1, BQ425406.1, BF029347.1, CD557751.1, BU507911.1,BF977553.1, BF976962.1, BF700934.1, BF219086.1, BF244797.1, BF700668.1,BF246890.1, BF684028.1, BF667950.1, BF666100.1, BF665251.1, AU151690.2,BF540908.1, AU099103.1, BF576929.1, BG403284.1, BF589484.1, AL583281.3,AL583241.3, AL531915.3, AL531914.3, AL527360.3, BF984727.1, AL527307.3,BG257162.1, BF899120.1, BF994346.1, BF978548.1, BU633554.1, AA188980.1,AA188981.1, AA189047.1, AA211359.1, BU943322.1, AA249583.1, AA249666.1,CA311030.1, CA446863.1, AA312280.1, T89463.1, AA492580.1, AA628019.1,AA639709.1, AA700427.1, AA857356.1, AA878068.1, BG679506.1, BI087094.1,BI260526.1, N88235.1, BI861865.1, BM194061.1, BM453009.1, BM476696.1,BM806248.1, BM828736.1, BM995677.1, W72679.1, AL711170.1, AL710845.1,BQ307283.1, BQ307306.1, BQ776047.1, BQ776118.1, CN294243.1, CN294241.1,CN294240.1, CN294239.1, CN294238.1, CN294237.1, AW821289.1, AW955129.1,AW573107.1, AW449014.1, BP430527.1, AW069561.1, AI979323.1, BX453617.2,AI955047.1, AI954614.1, BX431751.2, BX372554.2, BX353230.2, BX351284.2,BX333981.2, AI913466.1, AI866885.1, BX284007.1, BF246242.1, AI660156.1,AI380253.1, BP369219.1, BF218992.1, BP282259.1, AU129601.1, BP283529.1,AI341287.1, BF084870.1, BP290528.1, BP242960.1, BP239153.1, CV030676.1,CN294245.1, CN294236.1, BF082771.1, BE503512.1, AI341285.1, BF038467.1,BE543964.1, CB150849.1, CB160181.1, BF001266.1, BE672102.1, CN294234.1,CN294233.1, R94766.1, CV363738.1, BE927464.1, AA911686.1, AV718407.2,BE940500.1, BE857720.1, BE889796.1, AV718036.1, BF687621.1, BF447144.1,CN294232.1, CN294235.1, CN294244.1, R92253.1

A preferred embodiment of the present invention provides anapoptosis-inducing agent comprising double-strand RNA having RNAieffects and having as one of the strands thereof a contiguous RNA regionof mRNA corresponding to a chromosome stabilization-associated gene (forexample, any of the aforementioned genes) or any of the aforementionedESTs.

As described above, each of the aforementioned genes may contain variouspolymorphisms even for the same gene. Those skilled in the art cansuitably design an RNA sequence expected to have RNAi effects for any ofthe nucleotide sequences described in SEQ ID NOs: 1 to 637 and 810 to908 or the aforementioned EST sequences by incorporating data from, forexample, a public polymorphism database relating to any of theaforementioned genes. An apoptosis-inducing agent comprising such an RNAis also included in the present invention. In addition, RNA havingoptimum RNAi effects can be suitably selected by those skilled in theart from several types of double-strand RNA produced in the presentinvention to obtain an apoptosis-inducing agent.

Specific examples of the aforementioned “double-strand RNA having RNAieffects” of the present invention include siRNA molecules having as oneof the strands of the double-strand RNA a nucleotide sequence describedin FIGS. 1 to 4 and FIGS. 28 to 32 (a nucleotide sequence described inany of SEQ ID NOs: 724 to 809 and 974 to 1063) (siRNA molecules composedof a nucleotide sequence described in any of SEQ ID NOs: 724 to 809 and974 to 1063, and a complementary strand thereto). Namely, an embodimentof the present invention provides a cancer cell-specificapoptosis-inducing agent comprising as its active ingredient an siRNAmolecule in which one of the strands of the double-strand RNA havingRNAi effects comprises a nucleotide sequence described in any of SEQ IDNOs: 724 to 809 and 974 to 1063 (an siRNA molecule composed of anucleotide sequence described in any of SEQ ID NOs: 724 to 809 and 974to 1063, and a complementary strand thereto).

In addition, one of the RNA sequences of a double strand region in theaforementioned siRNA molecule is not necessarily limited to that whichis completely identical to a nucleotide sequence described in any of theaforementioned SEQ ID NOs: 724 to 809 and 974 to 1063. For example, theaforementioned siRNA molecule may be an siRNA molecule having as one ofthe strands of the double-strand RNA a nucleotide sequence in which oneor more nucleotides in the nucleotide sequence have been altered, aslong as it has a function which inhibits expression of a gene of thepresent invention.

Namely, in a preferred embodiment of the present invention,double-strand RNA having RNAi effects is double-strand RNA having afunction which inhibits expression of a gene of the present invention,in which one of the strands of the double strand is a nucleotidesequence having one or more nucleotide additions, deletions orsubstitutions to a nucleotide sequence described in any of SEQ ID NOs:724 to 809 and 974 to 1063, and the other strand is a nucleotidesequence complementary to the nucleotide sequence. The above “more”usually refers to a small number, and more specifically, refers to 2 to10, preferably 2 to 5, and more preferably 2 to 3.

In addition, a preferred embodiment of the present invention provides anapoptosis-inducing agent in which the compound which inhibits expressionof a chromosome stabilization-associated gene (for example, any of theaforementioned genes) is (a) or (b) below:

(a) an antisense nucleic acid to a transcription product, or a portionthereof, of a chromosome stabilization-associated gene of the presentinvention (for example, any of the aforementioned genes), or

(b) a nucleic acid having ribozyme activity which specifically cleaves atranscription product of a chromosome stabilization-associated gene ofthe present invention (for example, any of the aforementioned genes).

As used herein, the term “nucleic acid” refers to RNA and DNA. Methodswell known to those skilled in the art for inhibiting (suppressing) theexpression of a specific endogenous gene include those using antisensetechnology. Multiple factors contribute to the inhibition of a targetgene expression by an antisense nucleic acid. These factors include, forexample, inhibition of transcription initiation through triplexformation; inhibition of transcription through hybrid formation with asequence at the site of a local open loop structure made by RNApolymerase; inhibition of transcription through hybrid formation withthe RNA being synthesized; inhibition of splicing through hybridformation with a sequence at an intron-exon junction; inhibition ofsplicing through hybrid formation with a sequence at the site ofspliceosome formation; inhibition of transfer from the nucleus to thecytoplasm through hybrid formation with mRNA; inhibition of splicingthrough hybrid formation with a sequence at the capping site or poly(A)site; inhibition of translation initiation through hybrid formation witha sequence at the site of binding of the translation initiation factor;inhibition of translation through hybrid formation with a sequence atthe ribosome binding site near the initiation codon; inhibition ofpeptide chain elongation through hybrid formation with a sequence at thesite of the translational region or polysome binding site of the mRNA;and inhibition of gene expression through hybrid formation with asequence at the site of interaction between the expression regulatoryregion and the transcriptional regulatory factor. Thus, an antisensenucleic acid inhibits target gene expression by inhibiting variousprocesses, such as transcription, splicing, and translation (Hirashimaand Inoue, Shin Seikagaku Jikkenkoza 2 (New Lecture for ExperimentalBiochemistry 2), Kakusan IV (Nucleic Acid IV), Replication andExpression of Genes; Ed., Japanese Biochemical Society, Tokyo KagakuDozin Co., Ltd., pp. 319-347, 1993).

Antisense nucleic acids used in the present invention may inhibit theexpression of a chromosome stabilization-associated gene (e.g., any ofthe aforementioned genes) through any one of the actions describedabove. In one embodiment, an antisense sequence is designed to becomplementary to the 5′-untranslated region of a chromosomestabilization-associated gene (e.g., any of the aforementioned genes)mRNA. Thus such an antisense sequence is expected to effectively inhibittranslation of that gene. A sequence complementary to the coding regionor 3′-untranslated region can also be used for this purpose. Thus, anucleic acid comprising the antisense sequence corresponding to thesequence of the translated as well as the untranslated regions of thechromosome stabilization-associated gene (e.g., any of theaforementioned genes) can be included as an antisense nucleic acid usedin the present invention. The antisense nucleic acid to be used isligated downstream of an appropriate promoter and preferably ligatedwith a sequence comprising a transcription termination signal at the 3′end. The antisense nucleic acid to be used for clinical applications istypically a synthetic oligomer. Such synthetic oligomers include thewidely used S-oligo (phosphorothioate oligo nucleotide) in which S(sulfur) has been substituted for O (oxygen) at the phosphate esterbond, thus reducing sensitivity to nuclease digestion and maintainingantisense nucleic acid activity. S-oligo is currently being tested as anantisense drug in clinical trials where it is administered directly toaffected areas. This S-oligo is also suitable for use in the presentinvention. It is preferable that the antisense nucleic acid sequence iscomplementary to the target gene sequence or a portion thereof; howeverperfect complementarity is not necessary as long as the antisensenucleic acid effectively suppresses target gene expression. Thetranscribed RNA has preferably 90% or higher complementarity, and mostpreferably 95% or higher complementarity to the target gene transcript.The length of the antisense nucleic acid used to effectively suppresstarget gene expression is at least 15 nucleotides or longer, preferably100 nucleotides or longer, and more preferably 500 nucleotides orlonger.

The inhibition of chromosome stabilization-associated gene (e.g., any ofthe aforementioned genes) expression can also be achieved using aribozyme or ribozyme-encoding DNA. The term “ribozyme” refers to an RNAmolecule comprising catalytic activity. Ribozymes can have a variety ofactivities, and can be designed to have the activity of cleaving RNA ina site-specific fashion. Ribozymes such as group I intron-type ribozymesand MI RNA, which are RNase P ribozymes, are 400 nucleotides or more inlength. Others such as hammerhead and hairpin ribozymes have activesites comprising about 40 nucleotides (M. Koizumi and E. Otsuka,Tanpakushitsu Kakusan Koso (Protein, Nucleic acid, and Enzyme), 1990,35, 2191).

For example, the autolytic domain of a hammerhead ribozyme cleaves the3′ side of C15 in the sequence G13U14C15. Base pairing between U14 andA9 plays an important role in this activity, and A15 or U15 can becleaved instead of C15 (Koizumi, M. et al., FEBS Lett, 228: 228, 1988).A restriction enzyme-like RNA-cleaving ribozyme that recognizes thetarget RNA sequences UC, UU, or UA can be produced by designing theribozyme such that the substrate binding site complements the RNAsequence near the target site (Koizumi, M. et al., FEBS Lett, 239: 285,1988; M. Koizumi and E. Otsuka, Tanpakushitsu Kakusan Koso (Protein,Nucleic acid, and Enzyme), 35:2191, 1990; and Koizumi, M. et al., Nucl.Acids Res., 17: 7059, 1989).

The hairpin ribozyme can also be used for the purposes of the presentinvention. This ribozyme is found, for example, in the minus strand oftobacco ring spot virus satellite RNA (Buzayan, J. M., Nature, 323: 349,1986). A target specific RNA-cleaving ribozyme can also be produced froma hairpin ribozyme (Kikuchi, Y. and Sasaki, N., Nucl. Acids Res., 19:6751, 1991; Kikuchi, H., Kagaku to Seibutsu (Chemistry and Biology),30:112, 1992). Thus, the expression of a chromosomestabilization-associated gene of the present invention can be inhibitedby specifically digesting the gene transcript using a ribozyme.

The present invention also relates to a cancer cell-specificapoptosis-inducing agent comprising as its active ingredient a compoundwhich inhibits the function (activity) of a protein encoded by achromosome stabilization-associated gene (for example, any of theaforementioned genes).

A protein encoded by a chromosome stabilization-associated gene of thepresent invention includes mutant proteins or homolog proteins of aprotein encoded by a chromosome stabilization-associated gene. Suchmutant proteins or homolog proteins are functionally equivalent to theprotein encoded by a chromosome stabilization-associated gene, and havean amino acid sequence with one or more amino acid deletions,substitutions, or additions to the amino acid sequence of the protein.Here, a “functionally equivalent protein” refers to a protein having afunction which is similar to the function (for example, any of thefunctions of the aforementioned (a) to (s)) of a protein encoded by achromosome stabilization-associated gene (for example, any of theaforementioned genes).

Alternatively, a protein having, for example, 90% or more, desirably 95%or more, and more desirably 99% or more homology with the amino acidsequence of a protein encoded by a chromosome stabilization-associatedgene (for example, any of the aforementioned genes) can be indicated asa protein functionally equivalent to a protein encoded by a chromosomestabilization-associated gene.

A preferred embodiment of the present invention provides anapoptosis-inducing agent in which a compound which inhibits the function(activity) of a protein encoded by a chromosome stabilization-associatedgene (for example, any of the aforementioned genes) is a compounddescribed in any of (a) to (c) below. These compounds are thought tohave an apoptosis-inducing action against cancer cells by inhibiting(decreasing) the function or activity of a protein encoded by achromosome stabilization-associated gene (for example, any of theaforementioned genes).

(a) Mutant proteins having dominant negative traits with respect to aprotein encoded by a chromosome stabilization-associated gene of thepresent invention (for example, any of the aforementioned genes).

(b) Antibodies which bind to a protein encoded by a chromosomestabilization-associated gene of the present invention (for example, anyof the aforementioned genes).

(c) Low molecular weight compounds which bind to a protein encoded by achromosome stabilization-associated gene of the present invention (forexample, any of the aforementioned genes).

The “mutant proteins having dominant negative traits” in above (a) referto mutants of a protein encoded by a chromosome stabilization-associatedgene (for example, any of the aforementioned genes) having a function todeactivate or decrease the activity of an endogenous wild-type protein.

The “antibodies” in above (b) can be prepared according to methods knownto those skilled in the art. Polyclonal antibodies, for example, can beobtained in the following manner. Serum is obtained from a small animalsuch as a rabbit immunized with a protein encoded by anaturally-occurring or recombinant chromosome stabilization-associatedgene (for example, any of the aforementioned genes) or a protein encodedby a recombinant chromosome stabilization-associated gene expressed inmicroorganisms such as Escherichia coli as a fusion protein with GST, ora partial peptide thereof. This serum is then purified by, for example,ammonium sulfate precipitation, protein A column and protein G column,DEAE ion exchange chromatography, or an affinity column coupled with aprotein or synthetic peptide encoded by a chromosomestabilization-associated gene. In addition, monoclonal antibodies can beprepared by, for example, immunizing a small animal such as a mouse witha protein, or a partial peptide thereof, encoded by a chromosomestabilization-associated gene (for example, any of the aforementionedgenes), excising the spleen from the mouse, gently grinding the excisedspleen to separate the cells, fusing the cells with mouse myeloma cellsusing a reagent such as polyethylene glycol, and selecting from theresulting fusion cells (hybridomas) those clones that produce anantibody which binds to the protein encoded by a chromosomestabilization-associated gene. Next, a hybridoma thus obtained istransplanted into the mouse abdominal cavity, peritoneal fluid isrecovered from the mouse. The resulting monoclonal antibody can then bepurified by, for example, ammonium sulfate precipitation, protein Acolumn and protein G column, DEAE ion exchange chromatography, or anaffinity column coupled with a protein or a synthetic peptide encoded bya chromosome stabilization-associated gene.

There are no particular restrictions on the antibody of the presentinvention so long as it is able to bind to a protein encoded by achromosome stabilization-associated gene of the present invention (forexample, any of the aforementioned genes). In addition to theaforementioned polyclonal antibody and monoclonal antibody, the antibodyincludes human antibodies, humanized antibodies obtained by geneticrecombination, and antibody fragments and antibody modification productsthereof.

There are no limitations on the animal species as the source of aprotein encoded by a chromosome stabilization-associated gene of thepresent invention (for example, any of the aforementioned genes), whichis used as a sensitizing antigen for acquiring antibody; however, aprotein of mammalian origin, such as that from a mouse or human, ispreferable, and a protein of human origin is particularly preferable.

Proteins to be used as a sensitizing antigen in the present inventionmay be intact proteins as well as partial peptides derived from thoseproteins. Such partial protein peptides include, for example, proteinamino (N)-terminal fragments and carboxyl (C)-terminal fragments. Asused herein, “antibody” usually refers to an antibody which reacts witha full-length protein or a fragment thereof.

In addition to obtaining the above-described hybridomas by immunizingnon-human animals with an antigen, hybridomas producing a desired humanantibody having binding activity with the protein can also be preparedin vitro by sensitizing human lymphocytes, for example, humanlymphocytes infected with EB virus, with the protein, cells expressingthe protein, or a lysate of those cells, and fusing these sensitizedlymphocytes with immortalized human myeloma cells, for example, U266cells. When an antibody of the present invention is intended to beadministered into human bodies (antibody therapy), a human antibody orhumanized antibody is preferable to reduce the immunogenicity.

Examples of compounds which are already known to bind to proteinsencoded by chromosome stabilization-associated genes include monoclonalor polyclonal antibodies directed to a protein encoded by any of theaforementioned genes.

Compounds which inhibit the expression of a chromosomestabilization-associated gene (for example, any of the aforementionedgenes) of the present invention or inhibit the function (activity) of aprotein encoded by the gene may be naturally-occurring or artificialcompounds. They are typically compounds which can be produced, obtained,or isolated using a method known to those skilled in the art. Examplesof such compounds include single compounds such as organic compounds,inorganic compounds, nucleic acids, proteins, peptides, and sugars, aswell as compound libraries, gene library expression products, cellextracts, cell culture supernatants, microbial fermentation products,marine organism extracts, plant extracts, and compounds isolated andpurified from the extracts.

The present invention also provides methods of screening for cancercell-specific apoptosis-inducing agents.

A preferred embodiment of the aforementioned methods of the presentinvention is a method which uses as an index the binding activitybetween a protein encoded by a chromosome stabilization-associated gene(for example, any of the aforementioned genes), or a partial peptidethereof, and a test compound. Normally, a compound which binds to aprotein encoded by a chromosome stabilization-associated gene, or apartial peptide thereof, is expected to have inhibitory effects on thefunction of a protein encoded by a chromosome stabilization-associatedgene (for example, any of the aforementioned genes).

In the aforementioned method of the present invention, a protein encodedby a chromosome stabilization-associated gene (for example, any of theaforementioned genes), or a partial peptide thereof, is first contactedwith a test compound. The protein encoded by a chromosomestabilization-associated gene, or partial peptide thereof, can be, forexample, in a purified form of the protein encoded by a chromosomestabilization-associated gene, or partial peptide thereof, or in a formexpressed within or outside cells, or in a form bound to an affinitycolumn, depending on the index for detecting its binding to the testcompound. Test compounds used in this method can be used after beingsuitably labeled as necessary. Examples of labels include radioactivelabels and fluorescent labels.

In the present method, the binding activity between the protein encodedby the chromosome stabilization-associated gene, or partial peptidethereof, and the test compound, is then measured. Binding activitybetween the protein encoded by the chromosome stabilization-associatedgene, or partial peptide thereof, and the test compound can be measuredby, for example, a label attached to the test compound bound to theprotein encoded by the chromosome stabilization-associated gene orpartial peptide thereof. In addition, binding activity can also bemeasured using as an index a change in the activity of the proteinencoded by the chromosome stabilization-associated gene expressed withinor outside cells, or partial peptide thereof, which occurs due tobinding of the test compound to the protein or partial peptide thereof.

In the present method, a test compound is then selected which binds to aprotein encoded by a chromosome stabilization-associated gene (forexample, any of the aforementioned genes), or partial peptide thereof.

There is no limitation as to the type of test compound used in thepresent invention. Such compounds include, but are not limited to, forexample, single unmixed compounds of organic compounds, inorganiccompounds, nucleic acids, proteins, peptides, sugars, natural compounds,and such; or compound libraries, expression products of gene libraries,cell extracts, cell culture supernatants, products of fermentingmicroorganisms, marine organism extracts, and plant extracts; andartificially synthesized compounds.

In an alternative embodiment of the screening method of the presentinvention, first, a test compound is contacted with cells that express achromosome stabilization-associated gene (for example, any of theaforementioned genes), or with a cell extract prepared from such cells.The phrase “cells that express a chromosome stabilization-associatedgene” described above includes cells expressing an endogenous chromosomestabilization-associated gene, and cells into which an exogenouschromosome stabilization-associated gene has been introduced and inwhich that gene is expressed. The cells in which an exogenous chromosomestabilization-associated gene is expressed can typically be prepared byintroducing into host cells an expression vector which contains thegene. Those skilled in the art can prepare such an expression vectorusing routine genetic engineering techniques. In the screening methodsof the present invention, cells expressing a chromosomestabilization-associated gene preferably include various tumor cells,for example, MCF7 (breast cancer), A549 (lung cancer), U2OS (osteogenicsarcoma), C33A (cervical cancer), HT1080 (fibrosarcoma), PA-1 (ovarianteratocarcinoma), Tera2 (embryonal carcinoma), T24 (bladder cancer),K562 (chronic myelocytic leukemia), Molt4 (acute lymphoblasticleukemia), AI72 (glioblastoma), HeLa (cervical cancer), HepG2 (hepaticcancer), ACC62 (melanoma), KP4 (pancreas cancer), CaKi-1 (kidneycancer), MKN45 (gastric cancer), LNcap (prostate cancer), MDA-MB435(breast cancer), EJ 1 (bladder cancer), and OVCAR3 (ovarian cancer).

Typically, but without limitation, a test compound is contacted withcells expressing a chromosome stabilization-associated gene by addingthe test compound to a culture medium of the cells expressing thechromosome stabilization-associated gene (for example, any of theaforementioned genes). When the test compound is a protein, the contactcan be achieved by introducing into the cells a DNA vector that allowsprotein expression.

The next step of this method comprises determining the expression levelof the chromosome stabilization-associated gene. Herein, the phrase“gene expression” refers to both transcription and translation. The geneexpression level can be determined using a method known to those skilledin the art. For example, mRNA can be extracted from cells expressing thechromosome stabilization-associated gene according to a conventionalmethod, and by using this mRNA as a template, the transcriptional levelof the gene can be determined using Northern hybridization or RT-PCR.Alternatively, the translational level of the gene can be determined bycollecting protein fractions from the cells expressing the chromosomestabilization-associated gene, and then detecting the expression of theprotein encoded by the gene using an electrophoresis method such assodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Furthermore, the translational level of the gene can be determined bydetecting the expression of the encoded protein by Western blottinganalysis using an antibody against the protein. There is no limitationas to the type of antibody used for detecting the protein encoded by thegene, as long as the protein can be detected. Such antibodies include,for example, both monoclonal and polyclonal antibodies.

In this method, a compound that it causes a reduction in expressionlevel when compared to the expression level measured in the absence of atest compound (control) is then selected. The compound selected by theabove-described procedure is expected to have the action of inducingapoptosis in cancer cells. This compound may be used as a carcinostatic(an anticancer agent) whose mode of action is based on apoptosisinduction.

In an alternative embodiment of the screening method of the presentinvention, a compound that reduces the expression level of a chromosomestabilization-associated gene (for example, any of the aforementionedgenes) of the present invention is selected using a reporter gene.

In this method, a test compound is first contacted with cells (or anextract of those cells) that comprise a DNA having a structure where areporter gene is operably linked to a transcriptional regulatory regionof a chromosome stabilization-associated gene (for example, any of theaforementioned genes). As used herein, the phrase “operably linked”means that the transcriptional regulatory region of the chromosomestabilization-associated gene is linked to a reporter gene in such a wayas to induce reporter gene expression when a transcriptional factorbinds to the transcriptional regulatory region of the gene. Thus, evenwhen the reporter gene is connected with another gene and thus forms afusion protein with that gene product, such a case is included in themeaning of “operably linked”, as long as the expression of the fusionprotein is induced when the transcriptional factor binds to thetranscriptional regulatory region of the gene. Using a known method andbased on the cDNA nucleotide sequence for a chromosomestabilization-associated gene (for example, any of the aforementionedgenes), those skilled in the art can obtain the transcriptionalregulatory region of that gene within the genome.

There is no limitation as to the type of reporter gene used in thismethod, as long as the expression of the reporter gene can be detected.Such reporter genes include, for example, the CAT gene, lacZ gene,luciferase gene, and GFP gene. The “cells that comprise a DNA having astructure where a reporter gene is operably linked to a transcriptionalregulatory region of a chromosome stabilization-associated gene”include, for example, cells into which a vector with a structure where areporter gene is operably linked to a transcriptional regulatory regionof a chromosome stabilization-associated gene (for example, any of theaforementioned genes) has been introduced. Those skilled in the art canprepare the above-described vector using routine genetic engineeringtechniques. The introduction of such a vector into cells can be achievedusing a conventional method, for example, using calcium phosphateprecipitation, electroporation, the lipofectamine method,microinjection, etc. “Cells that comprise a DNA having a structure wherea reporter gene is operably linked to a transcriptional regulatoryregion of a chromosome stabilization-associated gene” also includescells in which that structure has been inserted into the chromosome. ADNA structure can be inserted into a chromosome by using a methodroutinely used by those skilled in the art, for example, a randomintegration or gene transfer method using homologous recombination.

An “extract of cells that comprise a DNA having a structure where areporter gene is operably linked to a transcriptional regulatory regionof a chromosome stabilization-associated gene” includes, for example, amixture prepared by adding a DNA to a cell extract included in acommercially available in vitro transcription/translation kit, wherethat added DNA comprises a structure where a reporter gene is operablylinked to a transcriptional regulatory region of a chromosomestabilization-associated gene (for example, any of the aforementionedgenes).

In this method, the “contact” can be achieved by adding a test compoundinto a culture medium of “cells that comprise a DNA having a structurewhere a transcriptional regulatory region of a chromosomestabilization-associated gene is operably linked to a reporter gene”, orby adding a test compound into the above-described commerciallyavailable cell extract, which contains the DNA. However, the method ofcontact is not limited to the methods described above. When the testcompound is a protein, the contact can also be achieved, for example, byintroducing into the cells a DNA vector that directs the expression ofthe protein.

The next step of this method comprises determining the level of reportergene expression. The expression level of the reporter gene can bedetermined by a method that depends on the type of the reporter gene andwhich is known to those skilled in the art. For example, when thereporter gene is the CAT gene, expression level can be determined bydetecting the acetylation of chloramphenicol, mediated by the CAT geneproduct. When the reporter gene is the lacZ gene, expression level canbe determined by detecting color development in a chromogenic compound,mediated by the catalytic action of the lacZ gene expression product.When the reporter gene is the luciferase gene, the level can bedetermined by detecting the fluorescence of a fluorescent compound,mediated by the catalytic action of the luciferase gene expressionproduct. Alternatively, when the reporter gene is the GFP gene, thelevel can be determined by detecting the fluorescence of the GFPprotein.

The next step of this method comprises selecting compounds that reducereporter gene expression level as compared to expression leveldetermined in the absence of a test compound. The compounds selected bythe above-described procedure can be cancer cell-specific apoptosisinducing agents.

Another embodiment of the method of the present invention is a method ofscreening for compounds by using as an index the activity of a proteinencoded by a chromosome stabilization-associated gene (for example, anyof the aforementioned genes) of the present invention.

In this method, a protein encoded by a chromosomestabilization-associated gene (for example, any of the aforementionedgenes) or cells expressing the protein, or a cell extract thereof, isfirst contacted with a test compound. Next, the activity of the proteinis measured. Examples of the activity of the protein include thefunctions (activities) indicated in the aforementioned (a) to (r). Thoseskilled in the art are able to suitably acquire information on thefunctions (activities) of proteins used as indexes in screening andinformation on methods for evaluating (measuring) the functions(activities) from, for example, a reference database.

For example, when the protein used as an index is Mcm10, the function ofthe protein can be evaluated (measured) by detecting the behavior of ARS(autonomously replicating sequences) with two-dimensionalelectrophoresis (MCB (1997) 3261-3271).

When the protein used as an index is Orc1, the function of the proteincan be evaluated (measured) by, for example, detecting the formation ofa complex of Orc1-6 which contains the protein or by detecting a changein the electrophoretic mobility of an Orc1-6 complex in the presence ofCaCl₂ (JBC (1998) 273, 32421-32429).

When the protein used as an index is Orc3, the function of the proteincan be evaluated (measured) by, for example, detecting the formation ofa complex of Orc1-6 which contains the protein or by detectingOri-specific binding of the protein (JCB (1998) 273, 32421-32429).

When the protein used as an index is Cdc6, the function of the proteincan be evaluated (measured) by, for example, using a cell-free DNAreplication assay (EMBO (1998) 17, 7219-7229).

When the protein used as an index is Cdt1, the function of the proteincan be evaluated (measured) by, for example, detecting binding of theprotein with Geminin, or by using a cell-free DNA replication assay(Science (2000) 290, 2309-2312).

When the protein used as an index is Geminin, the function of theprotein can be evaluated (measured) by, for example, detecting DNAreplication inhibitory activity using a cell-free DNA replication assay(Cell (1998) 93, 1043-1053).

When the protein used as an index is Mcm3, the function of the proteincan be evaluated (measured) by, for example, detecting the activity ofan Mcm2,3,5 complex containing the protein which inhibits the helicaseactivity of Mcm4,6,7 (JBC (1998) 273, 8369-8375).

When the protein used as an index is Mcm4, the function of the proteincan be evaluated (measured) by, for example, detecting the ssDNA bindingactivity, ATPase activity, and helicase activity of an Mcm4,6,7 complexcontaining the protein (JBC (1997) 272, 24508-24513).

When the protein used as an index is Mcm5, the function of the proteincan be evaluated (measured) by, for example, detecting the inhibitoryactivity on the helicase activity of Mcm4,6,7 by an Mcm2,3,5 complexcontaining the protein (JBC (1998) 273, 8369-8375).

When the protein used as an index is Mcm6, the function of the proteincan be evaluated (measured) by, for example, detecting the ssDNA bindingactivity, ATPase activity, and helicase activity of an Mcm4,6,7 complexcontaining the protein (JBC (1997) 272, 24508-24513).

When the protein used as an index is Mcm7, the function of the proteincan be evaluated (measured) by, for example, detecting the ssDNA bindingactivity, ATPase activity, and helicase activity of an Mcm4,6,7 complexcontaining the protein (JBC (1997) 272, 24508-24513).

When the protein used as an index is Mcm8, the function of the proteincan be evaluated (measured) by, for example, detecting binding betweenthe protein and an Mcm4,6,7 complex (Nucleic Acids Res. (2003) 31,570-579).

When the protein used as an index is Cdc7, the function of the proteincan be evaluated (measured) by, for example, detecting thephosphorylation activity of the protein using an MCM complex as thesubstrate (EMBO (1997) 16, 4340-4351).

When the protein used as an index is cdc5, the function of the proteincan be evaluated (measured) by, for example, detecting the transcriptionactivation ability of the protein (JBC (1998) 273, 4666-4671).

When the protein used as an index is Psf1, the function of the proteincan be evaluated (measured) by, for example, detecting the formation ofa GINS complex between Psf1-4 containing the protein and Sld5, or bydetecting binding of Dpb11, Sld3, and Cdc47 to the Ori sequence by GINS(Genes & Dev. (2003) 17, 1153-1165).

When the protein used as an index is Psf2, the function of the proteincan be evaluated (measured) by, for example, detecting the formation ofa GINS complex between Psf1-4 containing the protein and Sld5, or bydetecting binding of Dpb11, Sld3, and Cdc47 to the Ori sequence by GINS(Genes & Dev. (2003) 17, 1153-1165).

When the protein used as an index is Psf3, the function of the proteincan be evaluated (measured) by, for example, detecting the formation ofa GINS complex between Psf14 containing the protein and Sld5, or bydetecting binding of Dpb11, Sld3, and Cdc47 to the Ori sequence by GINS(Genes & Dev. (2003) 17, 1153-1165).

When the protein used as an index is Cdc45, the function of the proteincan be evaluated (measured) by, for example, detecting binding of theprotein to Mcm7 and Pola p70 (Eur. J. Biochem. 265, 936-943).

When the protein used as an index is Pola p180, the function of theprotein can be evaluated (measured) by, for example, detecting theformation of a tetramer composed of Pola p180, p70, p58, and p48containing the protein, or by detecting the primase or polymeraseactivity of this complex (Eur. J. Biochem. 222, 781-793).

When the protein used as an index is Pola p70, the function of theprotein can be evaluated (measured) by, for example, detecting theformation of a tetramer composed of Pola p180, p70, p58, and p48containing the protein, or by detecting the primase or polymeraseactivity of this complex (Eur. J. Biochem. 222, 781-793).

When the protein used as an index is Pola Spp1 (p58), the function ofthe protein can be evaluated (measured) by, for example, detecting theformation of a tetramer composed of Pola p180, p70, p58, and p48containing the protein, or by detecting the primase or polymeraseactivity of this complex (Eur. J. Biochem. 222, 781-793).

When the protein used as an index is RPA70, the function of the proteincan be evaluated (measured) by, for example, detecting the binding ofthe protein to ssDNA (Nature (1997) 385, 176-181).

When the protein used as an index is RPA34, the function of the proteincan be evaluated (measured) by, for example, detecting binding of theprotein to ssDNA or by using an in vitro replication assay including theprotein (JBC (1990) 265, 3177-3182).

When the protein used as an index is PCNA, the function of the proteincan be evaluated (measured) by, for example, using an in vitroreplication assay including the protein (JBC (1990) 265, 3177-3182).

When the protein used as an index is Ligase 1, the function of theprotein can be evaluated (measured) by, for example, the DNA ligationactivity involving the protein (PNAS (1990) 87, 6679-6683).

When the protein used as an index is Pole Pol2, the function of theprotein can be evaluated (measured) by, for example, detecting the DNAsynthesis activity of a Pole purified preparation containing the protein(PNAS (1990) 87, 6664-6668).

When the protein used as an index is Pole Dpb3, the function of theprotein can be evaluated (measured) by, for example, detecting the DNAsynthesis activity of a purified Pole preparation containing the protein(PNAS (1990) 87, 6664-6668).

When the protein used as an index is Topoisomerase 1, the function ofthe protein can be evaluated (measured) by, for example, detecting therelaxing activity of the protein using plasmid DNA as the substrate(PNAS (1988) 85, 2543-2547).

When the protein used as an index is TDP1, the function of the proteincan be evaluated (measured) by, for example, detecting the activity ofthe protein which liberates a tyrosine residue bound to the 3′ end ofssDNA (Science (1999) 286, 552-555).

When the protein used as an index is FEN1, the function of the proteincan be evaluated (measured) by, for example, detecting the flapstructure removal activity using as the substrate double-strand DNAhaving a 5′-overhanging flap structure (Genomics (1995) 25, 220-225).

When the protein used as an index is Pold P125, the function of theprotein can be evaluated (measured) by, for example, detecting theformation of a heterotetramer by the protein and Pold P68, P50, and P12(Biochemistry 2002 41(44): 13133-13142).

When the protein used as an index is Pole Dpb4, the function of theprotein can be evaluated (measured) by, for example, detecting the DNAsynthesis activity of a purified Pole preparation containing the protein(PNAS (1990) 87, 6664-6668).

When the protein used as an index is DNA2, the function of the proteincan be evaluated (measured) by, for example, detecting ssDNA bindingability and ATPase activity (PNAS (1995) 92, 7642-7646).

When the protein used as an index is ATR, the function of the proteincan be evaluated (measured) by, for example, binding the protein todouble-strand DNA having a UV-damaged site, or by detectingphosphorylation by the protein using p53 protein as the substrate (PNAS(2002) 99, 6673-6678).

When the protein used as an index is Chk1, the function of the proteincan be evaluated (measured) by, for example, detecting phosphorylationby the protein using p53 protein as the substrate (Genes Dev. (2000) 14,289-300).

When the protein used as an index is NBS1, the function of the proteincan be evaluated (measured) by, for example, detecting the formation ofa complex of the protein with Mre11/Rad50 in response to DNA damage(Cell (1998) 93, 477-486).

When the protein used as an index is Hus1, the function of the proteincan be evaluated (measured) by, for example, detecting the formation ofa complex of the protein with Rad1 and Rad9 in response to DNA damage(JCB (1999) 274, 567-570).

When the protein used as an index is Rad1, the function of the proteincan be evaluated (measured) by, for example, detecting the formation ofa complex of the protein with Hus1 and Rad9 in response to DNA damage(JBC (1999) 274, 567-570).

When the protein used as an index is Mad2, the function of the proteincan be evaluated (measured) by, for example, detecting the formation ofa complex of the protein and Mad1 (Science 274 (1996) 246-248).

When the protein used as an index is Ctf18, the function of the proteincan be evaluated (measured) by, for example, detecting the formation ofa complex of the protein with Ctf8 and Dcc1 (JBC (2003) 30051-30056).

When the protein used as an index is Scc3, the function of the proteincan be evaluated (measured) by, for example, detecting the formation ofa 14S cohesin complex of the protein with Smc1, Smc3, and Scc1 (JCB(2000) 151, 749-761).

When the protein used as an index is Scc3, the function of the proteincan be evaluated (measured) by, for example, detecting the formation ofa 14S cohesin complex of the protein with Smc1, Smc3, and Scc1 (JCB(2000) 151, 749-761).

When the protein used as an index is UNG, the function of the proteincan be evaluated (measured) by, for example, detecting glycosylaseactivity using deoxyuridine in ssDNA as the substrate (EMBO (1989) 8,3121-3125).

When the protein used as an index is MBD4, the function of the proteincan be evaluated (measured) by, for example, detecting the bindingactivity of the protein to a methylated CpG sequence (MCB (1998) 18,6538-6547).

When the protein used as an index is NTH1, the function of the proteincan be evaluated (measured) by, for example, detecting the glycosylaseactivity and AP lyase activity of the protein (PNAS (1997) 94, 109-114).

When the protein used as an index is NEIL2, the function of the proteincan be evaluated (measured) by, for example, detecting the AP lyaseactivity of the protein using DNA having a damaged base as the substrate(JBC (2002) 277, 30417-30420).

When the protein used as an index is NEIL3, the function of the proteincan be evaluated (measured) by, for example, detecting the AP lyaseactivity of the protein using DNA containing an 8-oxo, AP site, and5-hydroxycytosine as the substrate (Nucleic Acids Res. (2002) 316,853-866).

When the protein used as an index is APE2, the function of the proteincan be evaluated (measured) by, for example, detecting the APendonuclease activity of the protein (JMB (2002) 316, 853-866).

When the protein used as an index is PARP1, the function of the proteincan be evaluated (measured) by, for example, detecting the ADP-ribosepolymerase activity of the protein on nicked DNA using ADP-ribose as thesubstrate (JBC (1990) 35, 21907-21913).

When the protein used as an index is PNK, the function of the proteincan be evaluated (measured) by, for example, detecting thepolynucleotide kinase activity using oligo(dT) as the substrate (JBC(1999) 274, 24176-24186).

When the protein used as an index is Polb, the function of the proteincan be evaluated (measured) by, for example, detecting the gap-fillingpolymerase activity of the protein (Biochemistry (1988) 901-909).

When the protein used as an index is MGMT, the function of the proteincan be evaluated (measured) by, for example, detecting a reaction inwhich a methyl group is transferred from methylated DNA by the protein(JBC (1990) 265, 14754-14762).

When the protein used as an index is TDG, the function of the proteincan be evaluated (measured) by, for example, detecting the mismatchedthymidine-cleaving activity of the protein (JBC (1993) 268,21218-21224).

When the protein used as an index is MSH2, the function of the proteincan be evaluated (measured) by, for example, detecting binding of theprotein to double-strand DNA containing a mismatch (Cancer Res. (1994)54, 5539-5542).

When the protein used as an index is PMS1, the function of the proteincan be evaluated (measured) by, for example, detecting DNA bindingability and ATPase activity of the protein (Nucleic Acids Res. (2003)31, 2025-2034).

When the protein used as an index is PMS2, the function of the proteincan be evaluated (measured) by, for example, detecting the interactionof the protein with MLH1 (Hum. Mutat. 19, 108-113).

When the protein used as an index is Exonuclease 1, the function of theprotein can be evaluated (measured) by, for example, detecting theexonuclease activity of the protein (Nucleic Acids Res. (1998) 26,3762-3768).

When the protein used as an index is XPC, the function of the proteincan be evaluated (measured) by, for example, detecting the bindingability of the protein to ssDNA (EMBO (1994) 15, 1831-1843).

When the protein used as an index is Rad23A, the function of the proteincan be evaluated (measured) by, for example, detecting the interactionof its N terminal with the 26S proteasome and binding of its C terminalwith Rad4 (Nature (1998) 391, 715-718).

When the protein used as an index is Rad23B, the function of the proteincan be evaluated (measured) by, for example, detecting the interactionof its N terminal with the 26S proteasome and binding of its C terminalwith Rad4 (Nature (1998) 391, 715-718).

When the protein used as an index is CSA, the function of the proteincan be evaluated (measured) by, for example, detecting the interactionof the protein with CSB and TFIIH (Cell (1995) 82, 555-564).

When the protein used as an index is CSB, the function of the proteincan be evaluated (measured) by, for example, detecting the DNA-dependentATPase activity of the protein (JBC (1997) 272, 1885-1890).

When the protein used as an index is XPG, the function of the proteincan be evaluated (measured) by, for example, detecting the endonucleaseactivity of the protein using a partial duplex having a bubble structureas the substrate (Nature (1994) 371, 423-425).

When the protein used as an index is XPF, the function of the proteincan be evaluated (measured) by, for example, detecting the formation ofa complex between the protein and ERCC1, and the endonuclease activityof the protein using DNA having a stem-loop structure as the substrate(Cell (1996) 86, 811-822).

When the protein used as an index is DDB1, the function of the proteincan be evaluated (measured) by, for example, detecting the binding ofthe protein to UV-irradiated DNA (JBC (1993) 268, 21293-21300).

When the protein used as an index is XAB2, the function of the proteincan be evaluated (measured) by, for example, detecting the interactionof the protein with XPA, CSA, CSB, and RNA polymerase II (JBC (2000)275, 34931-34937).

When the protein used as an index is DDB2, the function of the proteincan be evaluated (measured) by, for example, detecting the bindingactivity of the protein to UV-damaged DNA (DNA Repair (2002) 6,601-616).

When the protein used as an index is Topoisomerase IIIb, the function ofthe protein can be evaluated (measured) by, for example, detecting theinteraction with RecQ5 helicase (Nucleic Acids Res. (2000) 28,1647-1655).

When the protein used as an index is Rad51, the function of the proteincan be evaluated (measured) by, for example, detecting thessDNA-dependent ATPase activity of the protein (JBC (2002) 277,14417-14425).

When the protein used as an index is Rad51D, the function of the proteincan be evaluated (measured) by, for example, detecting the ssDNA-bindingability of a Rad51B/Rad51C/Rad51D/Xrcc2 complex containing the protein,and detecting the ssDNA-dependent ATPase activity (Genes Dev. (2001) 15,329-3307).

When the protein used as an index is XRCC2, the function of the proteincan be evaluated (measured) by, for example, detecting the ssDNA-bindingability of a Rad51B/Rad51C/Rad51D/Xrcc2 complex containing the protein,and the ssDNA-dependent ATPase activity (Nature (1999) 401, 397-399).

When the protein used as an index is Rad54, the function of the proteincan be evaluated (measured) by, for example, detecting the DNA-dependentATPase activity of the protein (Curr. Biol (1996) 6, 828-838).

When the protein used as an index is BRCA1, the function of the proteincan be evaluated (measured) by, for example, detecting the E3 ubiquitinligase activity of the protein (EMBO J. (2002) 21, 6755-6762).

When the protein used as an index is Ku80, the function of the proteincan be evaluated (measured) by, for example, detecting the binding ofthe protein to a DNA terminal and the formation of a complex with Ku70(PNAS (1990) 87, 1777-1781).

When the protein used as an index is XRCC4, the function of the proteincan be evaluated (measured) by, for example, detecting binding of theprotein to Ligase4 and the DNA binding of the protein (Cell (1995) 83,1079-1089).

When the protein used as an index is Ubc13, the function of the proteincan be evaluated (measured) by, for example, detecting the ubiquitinconjugating activity of the protein (Cell (1999) 96, 645-653).

When the protein used as an index is Rad6A, the function of the proteincan be evaluated (measured) by, for example, detecting the formation ofa complex of the protein with Rad18 (PNAS (1991) 88, 8865-8869).

When the protein used as an index is Rad18, the function of the proteincan be evaluated (measured) by, for example, detecting DNA binding ofthe protein (Nucleic Acids Res. (2000) 28, 2847-2854).

When the protein used as an index is FBH1, the function of the proteincan be evaluated (measured) by, for example, detecting the helicaseactivity of the protein (JCB (2002) 277, 24530-24537).

When the protein used as an index is Poli, the function of the proteincan be evaluated (measured) by, for example, detecting the activity ofcarrying out primer extension from mismatched partial duplex DNA (JBC(2001) 276, 30615-30622).

When the protein used as an index is DUT1, the function of the proteincan be evaluated (measured) by, for example, detecting the dUTPaseactivity of the protein (J. Biol. Chem. (1996) 271, 7745-7751).

When the protein used as an index is Tin2, the function of the proteincan be evaluated (measured) by, for example, detecting the interactionbetween the protein and TRF1 (Nat Genet (1999) 23, 405-412).

When the protein used as an index is Sir2, the function of the proteincan be evaluated (measured) by, for example, a histone deacetylationassay for the protein (Gene (1999) 234, 161-168).

When the protein used as an index is Elg1, the function of the proteincan be evaluated by, for example, detecting the formation of a complexof the protein with RFC2, RFC3, RFC4, and RFC5 (EMBO J. (2003) 22,4304-4313).

When the protein used as an index is TIMELESS, the function of theprotein can be evaluated (measured) by, for example, detecting theformation of a complex of the protein with mammalian clock periodproteins (mPERs) (Science (2003) 302, 439-442).

When the protein used as an index is Pif1, the function of the proteincan be evaluated (measured) by, for example, detecting the ATP-dependenthelicase activity and DNA-dependent ATPase activity of the protein.

When the protein used as an index is Mms4, the function of the proteincan be evaluated (measured) by, for example, detecting the endonucleaseactivity of a complex of the protein with Mus81 protein (JBC (2003) 278,21715-21720).

When the protein used as an index is Topoisomerase IIIa, the function ofthe protein can be evaluated (measured) by, for example, detecting thetopoisomerase activity of the protein (Nucleic Acids Res. (2002) 30,4823-4829).

When the protein used as an index is Mus81, the function of the proteincan be evaluated (measured) by, for example, detecting the endonucleaseactivity of a complex of the protein with Mms4 protein (JBC (2003) 278,21715-21720).

When the protein used as an index is SIRT1, the function of the proteincan be evaluated (measured) by, for example, detecting the NAD-dependenthistone deacetylase of the protein (Nature (2000) 403, 795-800).

When the protein used as an index is ESP1, the function of the proteincan be evaluated (measured) by, for example, detecting the proteaseactivity of the protein (FEBS Lett. (2002) 528, 246-250).

When the protein used as an index is MPG, the function of the proteincan be evaluated (measured) by, for example, detecting the glycosylaseactivity of the protein (Carcinogenesis (1996) 17, 2177-2182).

When the protein used as an index is Poll, the function of the proteincan be evaluated (measured) by, for example, detecting the DNApolymerase activity of the protein (J Biol Chem. (2000) 275,31233-31238).

When the protein used as an index is Polm, the function of the proteincan be evaluated (measured) by, for example, detecting the DNApolymerase activity of the protein (J Biol Chem. (2002) 277,44582-44587).

When the protein used as an index is EndoV, the function of the proteincan be evaluated (measured) by, for example, detecting the endonucleaseactivity of the protein.

When the protein used as an index is KNTC2 (NDC80), the function of theprotein can be evaluated (measured) by, for example, detecting theformation of a complex of the protein with human Nuf2 protein (Mol BiolCell. (2005) 16, 519-531).

Next, a compound is selected which lowers the activity of a proteinencoded by a chromosome stabilization-associated gene as compared tothat measured in the absence of the test compound. Although a proteinencoded by the gene used in this method is preferably an unmutatedfull-length protein, it may be a protein in which a portion of the aminoacid sequence has been substituted and/or deleted so long as it hasactivity equivalent to that of the protein.

The present invention also provides anticancer agents (pharmaceuticalcompositions for treating cancers) which comprise as an activeingredient a cancer cell-specific apoptosis inducing agent of thepresent invention.

The present invention also provides methods for producing apoptosisinducing agents or anticancer agents as pharmaceutical compositions. Inthis method a compound for the cancer cell-specific apoptosis inducingagent is first selected using a screening method of the presentinvention. Then, the selected compound is combined with apharmaceutically acceptable carrier. Such a pharmaceutically acceptablecarrier can include, but is not limited to, for example, detergents,excipients, coloring agents, flavoring agents, preservatives,stabilizers, buffers, suspensions, isotonizing agents, binders,disintegrating agents, lubricants, fluidizing agents, and correctives.Other conventional carriers can be also used appropriately.

The agents such as apoptosis inducing agents and anticancer agents ofthe present invention can be formulated by adding the above-indicatedcarriers as required and according to conventional methods. Morespecifically, such carriers include: light anhydrous silicic acid,lactose, crystalline cellulose, mannitol, starch, carmellose calcium,carmellose sodium, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylacetaldiethylamino acetate, polyvinylpyrrolidone,gelatin, medium chain triglyceride, polyoxyethylene hydrogenated castoroil 60, saccharose, carboxymethyl cellulose, cornstarch, and inorganicsalts.

The dosage forms for the agents described above include, for example,oral forms, such as tablets, powders, pills, dispersing agents,granules, fine granules, soft and hard capsules, film-coated tablets,pellets, sublingual tablets, and pastes; and parenteral forms, such asinjections, suppositories, endermic liniments, ointments, plasters, andliquids for external use. Those skilled in the art can select theoptimal dosage form depending on the administration route, subject, andsuch. Viral vectors such as retrovirus, adenovirus, and Sendai virusvectors, and non-viral vectors such as liposomes, may be used tointroduce, into the living body, DNAs expressing proteins encoded bychromosome stabilization-associated genes (for example, theaforementioned genes), or DNAs expressing antisense RNAs, ribozymes, orsiRNAs that suppress chromosome stabilization-associated genes.Alternatively, non-viral vectors such as liposomes, polymer micelles, orcationic carriers, may be used to introduce, into the living body,synthetic antisense nucleic acids or synthetic siRNAs that suppresschromosome stabilization-associated genes. The introduction methodsinclude, for example, in-vivo and ex-vivo methods.

The present invention also includes pharmaceutical compositionscomprising the above-described apoptosis-inducing action.

Ultimately, the dose of an agent or pharmaceutical composition of thepresent invention can be appropriately determined by a physicianconsidering the dosage form, administration method, patient's age,weight, symptoms, etc.

The present invention also relates to methods for inducing apoptosis indesired cancer cells. A preferred embodiment of these methods is amethod for inducing apoptosis in cells in which one wishes to induceapoptosis (target cells), comprising a step of administering(contacting) an apoptosis-inducing agent of the present invention to thecells. For example, when the active ingredient of an apoptosis-inducingagent of the present invention is a nucleic acid, that ingredient (thenucleic acid) is preferably introduced into the target cells.

Moreover, the present invention relates to a method for treating cancercomprising a step of administering an apoptosis-inducing agent oranticancer agent of the present invention to an individual (e.g., cancerpatient).

The “individual” in the aforementioned treatment method normally refersto a cancer patient, and although there are no particular limitations,it is preferably a human. In general, administration to an individualcan be carried out by a method known to those skilled in the art,examples of which include intraarterial injection, intravenousinjection, and subcutaneous injection. Although the dosage variesdepending on the weight and age of the patient, administration method,and so on, a suitable dosage can be appropriately selected by thoseskilled in the art. In addition, if the compound can be encoded by DNA,gene therapy can also be carried out by incorporating the DNA in avector for gene therapy. Examples of vectors for gene therapy includeviral vectors such as retroviral vectors, adenoviral vectors, andadeno-associated viral vectors, and non-viral vectors such as liposomes.A desired DNA can be administered to a patient by an ex vivo method orin vivo method using such a vector. In addition, a nucleic acid of thepresent invention can also be administered directly to an individual.

The present invention also relates to the use of a compound thatinhibits chromosome stabilization (for example, a compound whichinhibits expression of a gene of the present invention, or inhibits thefunction of a protein encoded by the gene) for producing anapoptosis-inducing agent or anticancer agent.

All prior art documents cited in the present specification areincorporated herein by reference.

EXAMPLES

The present invention will be described in detail below with referenceto Examples, but is not to be construed as being limited thereto.

In the Examples, genes used as “chromosome stabilization-associatedgenes” are the following 97 genes:

APE2, ATR, BRCA1, Chk1, Cdc5, Cdc6, Cdc7, Cdc45, Cdt1, CSA, CSB, Ctf18,DDB1, DDB2, DNA2, DUT, Elg1, EndoV, Esp1, Exonuclease1, FBH1, FEN1,Geminin, Hus1, KNTC2 (NDC80), Ku80, Ligase1, Mad2, MBD4, Mcm3, Mcm4,Mcm5, Mcm6, Mcm7, Mcm8, Mcm10, MGMT, MLH3, Mms4, MPG, MSH2, Mus81, NBS1,NEIL2, NEIL3, NTH1, Orc1, Orc3, PARP1, PCNA, Pif1, PMS1, PMS2, PNK, Polap180, Pola p70, Pola Spp1 (Prim2a), Polb, Pold p125, Pole Dpb3, PoleDpb4, Pole Pol2, Poli, Poll, Polm, Psf1, Psf2, Psf3, Rad1, Rad18,Rad23A, Rad23B, Rad51, Rad51D, Rad54, Rad6A, RPA34, RPA70, Scc1, Scc3,Sir2, SIRT1 (Sirtuin), TDG, TDP1, TIMELESS, Tin2, Topoisomerase I,Topoisomerase IIIa, Topoisomerase IIIb, Ubc13, UNG, XAB2, XPC, XPF, XPG,Xrcc2, and XRCC4.

Example 1 Cell Culturing

HeLa (human cervical carcinoma cells) and TIG3 (normal diploidfibroblasts) cells were used as human cultured cells. These humancultured cells were cultured in Dulbecco's modified Eagle's mediumcontaining 10% fetal calf serum and 50 μg/ml gentamicin under conditionsof 37° C. and 5% CO₂.

Example 2 Study of Chromosome Stabilization-Associated Gene ExpressionInhibition's Effects on Cancer Cell Proliferation

siRNA was selected for each of the aforementioned genes for the purposeof studying the effects of chromosome stabilization-associate geneexpression inhibition on cancer cell proliferation. Synthesis of siRNAwas carried out at Qiagen (Tokyo) and Dhamacon, Inc. (Colorado, USA).

The siRNA sequences for the aforementioned genes are shown in the columnentitled “siRNA sequence” of FIGS. 1 to 4. Only the sense strands areshown in the Sequence Listing, and the corresponding antisense strandsare omitted. In addition, the “dTdT” sequence of each siRNA sequence isabbreviated as “TT” in the Sequence Listing.

These siRNAs were introduced into human cervical carcinoma HeLa cells.More specifically, HeLa cells were inoculated and grown in a 24-wellplate 24 hours prior to transfection of siRNA, and then transfection wasperformed at 20 to 50% confluence. Oligofectamine (Invitrogen) was usedas the transfection reagent, and transfection was carried out accordingto the attached manual. mRNA expression of each gene was quantified byTaqman PCR 48 hours after introduction.

More specifically, total RNA was extracted from the cells 48 hours aftertransfection of siRNA using the RNeasy Mini Kit (Qiagen). The ABI PRISM7000 Sequence Detection System (Applied Biosystems) was used forquantitative PCR RT-PCR primers and TaqMan probes for each of theaforementioned genes and β-actin gene were purchased from AppliedBiosystems. The TaqMan One-Step RT-PCR Master Mix Reagents Kit (AppliedBiosystems) was used as the RT-PCR reaction reagents, and RT-PCR wascarried out according to the attached manual. Comparativequantifications were carried out using β-actin as a standard.

The expression of each mRNA in cells to which each siRNA was introducedwas compared to a value of 100% representing the expression of each mRNAin cells to which the control RNA (NS) was introduced. The siRNA foreach gene was found to efficiently inhibit expression of each mRNA asshown in the column entitled “Inhibition of gene expression in HeLacells” of FIGS. 1 to 4.

Example 3 Survival Rates of HeLa Cells

The siRNA for each of the aforementioned genes selected in Example 2 wasrespectively introduced into HeLa cells followed by an investigation ofthe cell survival rates 4 days later by an MTT assay. The number ofviable cells 96 hours after introduction was measured using the viablecell measurement reagent SF (Nacalai Tesque).

As a result, prominent decreases in the survival rates were observed inHeLa cells to which siRNA of each of the aforementioned genes wasintroduced, as shown in the column entitled “MTT assay (HeLa cells)” ofFIGS. 1 to 4.

Example 4 Apoptosis-Inducing Effects of siRNA in HeLa Cells

An investigation was made as to whether or not the decreases in survivalrates in HeLa cells into which siRNA for each of the aforementionedgenes was introduced occurred due to apoptosis. siRNA for each gene wasintroduced into HeLa cells, and apoptosis induction in the HeLa cells 48hours after introduction was studied using the TUNEL method.

As a result, apoptosis was observed to be prominently induced in allHeLa cells to which siRNA for each of the aforementioned genes wasintroduced, as shown in the column entitled “TUNEL method” of FIGS. 1 to4 and in the photographs of FIGS. 5 to 9. On the other hand, inductionof apoptosis was not observed in HeLa cells to which the control RNA(NS) was introduced (upper left panel “Non-specific” in FIG. 5).

Namely, it was revealed that effective induction of apoptosis occurs asa result of inhibiting the expression of each of the aforementionedgenes of the present invention.

Example 5 Effects of siRNA on Normal Cell Proliferation

A study was conducted on the effects of siRNA for each of theaforementioned genes on the proliferation of normal cells, human fetallung-derived diploid fibroblast TIG3 cells. Lipofectamine 2000(Invitrogen) was used as the transfection reagent, and siRNA for each ofthe aforementioned genes was respectively introduced into TIG3 cellsfollowed by measurement of mRNA expression of each gene by Taqman PCR 48hours after introduction. In this experiment, expression of each mRNA inTIG3 cells into which each siRNA was introduced was compared to a valueof 100% representing the expression of each mRNA in TIG3 cells intowhich control RNA (NS) was introduced.

As a result, mRNA expression of each of the aforementioned genes in TIG3cells to which each siRNA was introduced was inhibited considerably ascompared with expression of these mRNA in TIG3 cells to which thecontrol RNA (NS) was introduced, as shown in the column entitled“Inhibition of gene expression in TIG3 cells” of FIGS. 1 to 4.

Example 6 Survival Rates of TIG3 Cells

The aforementioned siRNAs were respectively introduced into TIG3 cellsfollowed by an investigation of the cell survival rates 4 days later byMTT assay. As a result, the survival rates of TIG3 cells to which siRNAfor each of the aforementioned genes was introduced were comparativelyhigher than the survival rates of HeLa cells to which the same siRNA wasintroduced, and there were no prominent decreases in survival ratesobserved, as shown in the column entitled “MTT assay (TIG3 cells)” ofFIGS. 1 to 4.

From these results, it is thought that apoptosis is induced cancercell-specifically through the inhibition of the expression of genes ofthe present invention.

Example 7 Analysis of Genome Breakdown Process Using Anti-Single-StrandDNA Antibody

Anti-single-strand DNA (anti-ssDNA) antibody is an antibody whichspecifically recognizes single-strand DNA. It is said that if thegenomic structure of DNA, which are originally composed of doublestrands, is broken down due to a chromosome destabilization such as DNAdamage, a single strand region will be partially exposed. Thus, the useof this antibody makes it possible to specifically recognize andvisualize this genome breakdown process.

HeLa cells were inoculated on a slide glass and transfected with siRNAfor each of the aforementioned genes. The cells were then fixed informalin about 30 hours after siRNA introduction, and reacted withanti-ssDNA antibody as the primary antibody. The cells were thenobserved with a confocal laser microscope using a fluorescent-labeledantibody against the anti-ssDNA antibody as the secondary antibody. As aresult, nuclei having single-strand DNA were stained green as shown inFIGS. 10 to 27.

Namely, DNA damage including single-strand DNA formation was confirmedto occur due to inhibition of expression of each of the aforementionedgenes.

Example 8 Cell Culturing

The 11 genes indicated below were used as “chromosomestabilization-associated genes” in the following Examples.

Pif1, Mms4, Topoisomerase IIIa, Mus81, SIRT1 (Sirtuin), Esp1, MPG, Poll,Polm, EndoV, and KNTC2 (NDC80)

In addition to the HeLa cells and TIG3 cells described in Example 1,normal human skin-derived diploid fibroblasts (HDF cells) were used ashuman cultured cells. Culturing was carried out under the sameconditions as Example 1.

Example 9 Study of Chromosome Stabilization-Associated Gene ExpressionInhibition's Effects on Cancer Cell Proliferation

siRNA for each of the aforementioned genes was selected for the purposeof studying the effects of inhibition of the expression of theaforementioned 11 chromosome stabilization-associated genes onproliferation of cancer cells. siRNA synthesis was carried out in thesame manner as Example 2.

The siRNA sequences of the aforementioned 11 genes are shown in thecolumn entitled “siRNA sequence” of FIGS. 28 to 32. Only the sensestrands are shown in the Sequence Listing, and the correspondingantisense strands are omitted.

These siRNAs were introduced into HeLa cells, specifically under thesame conditions as described in Example 2. mRNA expression of theaforementioned 11 genes was quantified by Taqman PCR 48 hours afterintroduction. Quantification was carried out using the same method asExample 2.

The expression of each mRNA in cells to which each siRNA was introducedwas compared to a value of 100% representing the expression of each mRNAin cells to which the control RNA (NS) was introduced. The siRNA foreach gene was found to have efficiently inhibited expression of eachmRNA as shown in the column entitled “Inhibition of gene expression in40 nM HeLa cells” of FIGS. 28 to 30, the column entitled “mRNAExpression” in HeLa cells of FIG. 31, or the column entitled“Expression” in HeLa cells of FIG. 32.

Example 10 Survival Rate of HeLa Cells

The siRNA for each of the aforementioned 11 genes was respectivelyintroduced into HeLa cells followed by investigation of the cellsurvival rates by MTT assay 4 days after introduction. The number ofviable cells at 96 hours after introduction was measured using viablecell measurement reagent SF (Nacalai Tesque).

As a result, prominent decreases in survival rates were observed in HeLacells to which siRNA for each of the aforementioned genes wasintroduced, as indicated in the column entitled “Inhibition ofproliferation in 40 nM HeLa cells” of FIGS. 28 to 30, the columnentitled “Inhibition of proliferation” in HeLa cells of FIG. 31, or thecolumn entitled “Proliferation” in HeLa cells of FIG. 32.

Example 11 Apoptosis-Inducing Effects of siRNA in HeLa Cells

An investigation was made as to whether or not the decreases in survivalrates in HeLa cells to which siRNA for each of the aforementioned 11genes was introduced occurred due to apoptosis. siRNA for each gene wasintroduced into HeLa cells, and apoptosis induction in the HeLa cells 48hours after introduction was studied using the TUNEL method.

As a result, apoptosis was observed to be prominently induced in allHeLa cells to which siRNA for each of the aforementioned genes wasintroduced, as shown in the column entitled “Apoptosis” in HeLa cells ofFIG. 31, the column entitled “Apoptosis” in HeLa cells of FIG. 32, thephotographs entitled “HeLa cells” of FIGS. 33 and 34, and the photographof TUNEL staining of HeLa cells of FIG. 35.

Namely, it was clarified that effective induction of apoptosis occurs asa result of inhibiting the expression of each of the aforementionedgenes of the present invention.

Example 12 Effects of siRNA on Normal Cell Proliferation

A study was conducted on the effects of siRNA for each of theaforementioned 11 genes on the proliferation of normal cells, humanfetal lung-derived diploid fibroblast TIG3 cells or human skinfibroblast HDF cells. Lipofectamine 2000 (Invitrogen) was used as thetransfection reagent, and siRNA for each of the aforementioned genes wasrespectively introduced into the TIG3 cells or HDF cells followed bymeasurement of mRNA expression of each gene by Taqman PCR 48 hours afterintroduction. In this experiment, expression of each mRNA in TIG3 cellsor HDF cells to which each siRNA was introduced was compared to a valueof 100% representing the expression of each mRNA in TIG3 cells or HDFcells to which control RNA (NS) was introduced.

As a result, mRNA expression of each of the aforementioned genes in TIG3cells or HDF cells to which each siRNA was introduced was inhibitedconsiderably as compared with expression of these mRNA in TIG3 cells orHDF cells to which the control RNA (NS) was introduced, as shown in thecolumn entitled “Inhibition of gene expression in 40 nM TIG3 cells” ofFIGS. 28 to 30, the column entitled “mRNA Expression” in HDF cells ofFIG. 31, or the column entitled “Expression” in HDF cells of FIG. 32.

Example 13 Survival Rates of TIG3 Cells and HDF Cells

siRNA for each of the aforementioned 11 genes was respectivelyintroduced into TIG3 cells or HDF cells followed by an investigation ofthe cell survival rates 4 days later by MTT assay. As a result, thesurvival rates of TIG3 cells or HDF cells to which siRNA for each of theaforementioned genes was introduced were comparatively higher than thesurvival rates of HeLa cells to which the same siRNA was introduced, andthere were no prominent decreases in survival rates observed, as shownin the column entitled “Inhibition of proliferation in 40 nM TIG3 cells”of FIGS. 28 to 30, the column entitled “Inhibition of proliferation” inHDF cells of FIG. 31, or the column entitled “Proliferation” in HDFcells of FIG. 32.

From these results, it is thought that apoptosis is cancercell-specifically induced through the inhibition of the expression ofgenes of the present invention.

Example 14 Analysis of Genome Breakdown Process Using Anti-Single-StrandDNA Antibody

HeLa cells were inoculated onto a slide glass and transfected with siRNAfor each of the genes of Pif1, Mms4, Topoisomerase IIIa, Mus81, SIRT1(Sirtuin), Esp1, MPG, Poll, Polm, and EndoV. The cells were then fixedin formalin for about 30 hours after introduction of siRNA, and reactedwith anti-ssDNA antibody as the primary antibody. The cells were thenobserved with a confocal laser microscope using a fluorescent-labeledantibody against the anti-ssDNA antibody as the secondary antibody. As aresult, nuclei having single-strand DNA were stained green as shown inFIG. 36.

Namely, DNA damage including single-strand DNA formation was confirmedto occur due to inhibition of expression of each of the aforementionedgenes.

INDUSTRIAL APPLICABILITY

The present invention's compounds which inhibit chromosome stabilizationin cells or compounds which inhibit the function of a chromosomestabilization-associated gene have an action to induce cancercell-specific apoptosis. Pharmaceutical compositions comprising suchcompounds are believed to become anticancer agents havingapoptosis-induction as the mechanism of action, while also having fewadverse side effects. The present invention provides, for the firsttime, cancer cell-specific anticancer agents which haveapoptosis-induction as the mechanism of action and which targetchromosome stabilization-associated genes.

Even if certain compounds are found to have an apoptosis-inducingaction, it is difficult to use the compounds as pharmaceuticals whentheir apoptosis-inducing actions in normal cells are unknown. This isbecause there may be a risk of adverse effects when the compounds haveapoptosis-inducing actions in normal cells. In other words, if thecompounds have apoptosis-inducing actions not specific to cancer cells,in general, it is practically difficult to use the compounds aspharmaceuticals. Accordingly, the agents (the compounds) of the presentinvention are very practical and effective because theirapoptosis-inducing actions are specific to cancer cells.

The mechanism by which apoptosis is induced cancer cell-specifically byinhibition of chromosome stabilization can be explained in the followingmanner based on findings of the present inventors.

Numerous cancer cells are known to have mutations or deletions in thecancer suppressor gene p53. In addition, oncogenesis is known to takeplace in some cases due to the occurrence of an abnormality in a DNAdamage checkpoint mechanism. If the expression of functional chromosomestabilizing genes is inhibited by siRNA or the like and their functionsare blocked in cancer cells having an abnormality in p53 or a DNA damagecheckpoint mechanism, the chromosome stabilization mechanism will failand it would no longer be possible to repair chromosomal DNA. In suchcells, it is thought that apoptosis will be induced due to the residualDNA damage that has not been repaired. On the other hand, it is thoughtthat in normal cells such as diploid fibroblasts, if the expression offunctional chromosome stabilizing genes is inhibited by siRNA or thelike and their functions are blocked, the cell cycle will be temporarilyinterrupted by the action of p53 and the DNA damage checkpointmechanism, thereby enabling damage in chromosomal DNA to be repaired.

In addition to providing cancer cell-specific apoptosis-inducing agents,the present invention provides extremely useful academic findings forelucidating the mechanism of cancer cell-specific apoptosis induction.

1. A cancer cell-specific apoptosis-inducing agent, comprising as anactive ingredient a double-strand RNA that inhibits expression of theKNTC2 (NDC80) gene, wherein the double-strand RNA comprises: a sense RNAconsisting of the sequence selected from the group consisting of SEQ IDNOs: 1005 to 1010, 1012 and 1017; and an antisense RNA consisting of asequence complementary to said sense RNA.
 2. A cancer cell-specificapoptosis-inducing agent, comprising as an active ingredient a DNAencoding a double-strand RNA comprising a sense RNA consisting of thesequence selected from the group consisting of SEQ ID NOs: 1005 to 1010,1012 and 1017; and an antisense RNA consisting of a sequencecomplementary to said sense RNA.
 3. An anticancer agent, comprising asan active ingredient an apoptosis-inducing agent of claim 1 or claim 2.4. A double-strand RNA comprising: a sense RNA consisting of thesequence selected from the group consisting of SEQ ID NOs: 1005 to 1010,1012 and 1017; and an antisense RNA consisting of a sequencecomplementary to said sense RNA.
 5. A vector comprising a DNA encodingthe double-strand RNA of claim 4.